Pharmaceutical Microbiology Laboratory 

(Pharmaceutical Microbiology)

B. Pharm, Second Year, Third Semester

          Course Objectives of Pharmaceutical Microbiology

Pharmaceutical  Microbiology will provide practical-based knowledge on microbiology and its application to Pharmaceutical preparation for quality analysis.

Course Contents of  Pharmaceutical Microbiology

1. Instrumentation in Microbiology Laboratory
2. Preparation of different media
3. Nutrient agar and both
4. Soybean casein digest broth
5. Sabouraud dextrose agar
6. Identification of Microorganism by different staining techniques (Gram’s staining, LPCB mount)
7. Bioburden determination of different substances used in pharmaceuticals
8. Microbial monitoring of the environment of pharmaceutical industries
9. Water quality testing
10. Air quality evaluation
11. Floor evaluation
12. Validation of autoclave, hot air oven, and incubator
13. Sterility testing of different pharmaceutical products-Infusion, Injection, and eye drops
14. Antibiotic Susceptibility Testing: Disk Diffusion methods
15. Determination of minimum inhibitory concentration (MIC and MBC) of antibiotics and other antimicrobial compounds by tube and agar diffusion methods: E- test
16. Testing efficacy of disinfectants and preservatives
17. Antigen-Antibody Reaction Based Tests

Instrumentation in Microbiology Laboratory

List of Instruments-

• Analytical /Electrical Balance
• Autoclave
• Bunsen burner
• Centrifuge
• Colony Counter
• Deep Freezer
• Homogenizer
• Hot plate
• Hot air oven
• Incubator
• Laminar Air Flow
• McFarland Densitometer
• Magnetic Stirrer
• Microscope
• pH Meter
• Refrigerator
• Spectrophotometer
• Vortex Mixture
• Water Bath
• Distillation plant

Among them, the most common instruments that you have to know in Pharmaceutical  Microbiology are-

Bunsen Burner

Introduction of Bunsen Burner

A Bunsen burner named after Robert Bunsen, the German chemist who introduced it in 1855 (from a design by Peter Desdega, who likely modified an earlier design by Michael Faraday), the Bunsen burner was the forerunner of the gas-stove burner and the gas furnace. It is a common piece of laboratory equipment that produces a single open gas flame, which is used for heating, sterilization, and combustion.

Parts of Bunsen burner

• Base Gas inlet: The gas inlet is a tubular projection below the air hole where the gas enters the Bunsen burner and mixes with the oxygen.
• Needle valve for gas flow adjustment
• Rotary barrel for air adjustment: The barrel is the main upright part of the Bunsen burner and the part where the flames come out. Never touch the barrel as it can get very hot while in use and can stay hot long after it has stopped being used.
• Airhole: The air hole is a coverable opening above the gas inlet of the barrel that allows air to enter the Bunsen burner, where it mixes with the gas.
  The air hole can be partially or completely covered by turning the collar.
• Collar: The collar is an adjustable metal tube that for covering or exposing the air hole. This controls how much oxygen can enter the Bunsen burner, and therefore how much oxygen can mix with the gas.
  The more oxygen that is allowed to enter the Bunsen burner, the hotter the flame will be. Always light the Bunsen burner with the air holes completely covered by the collar.
• Gas regulator: It helps to regulate follow of gas.
• Rubber tubing: It is a short section of tubing attached to the gas inlet that connects the Bunsen burner to the gas tap on your lab bench.
• Base: The base of the Bunsen burner is a flat disc that provides the support for it to stand up. It is also the safest part of the Bunsen burner to touch if you need to move or carry it, as it is designed not to get hot.

Procedure for Lighting a Bunsen Burner

1. Put on your safety goggles and lab apron. If you have long hair, make sure it is tied back.
2. Connect the rubber tubing to a gas tap.
3. Place a heat mat under the Bunsen burner if the surface area is not heat resistant.
4. Turn the collar so that the air hole is covered.
5. Light a match and hold it about 3 cm above the top of the barrel.
   Turn the gas tap to the ‘on’ position.
6. Once it is lit, extinguish the match.
7. Leave it’s flame in ‘safety mode’ until you need to heat something.

The temperature of Bunsen Burner

The amazing fact about the Bunsen burner is that the hottest part of the Bunsen flame which is heated just above the tip of the primary flame reaches about  1500 °C or 2700 °F. Having this type of high temperature and requiring less space, it is also called a micro incineration plant that is why also included in sterilization as a physical method.

Good and bad flame Recognition of Bunsen Burner

1. Good flame: Only blue in color
2. Bad flame : Blue with other colors

Uses of Bunsen Burner

1. Sterilization of Inoculating loop
2. Sterilization of stabbing straight wire
3. Sterilization of Forceps touched parts with specimens
4. A heating substance with help of a tripod stands using a safety flame.

Safety Rules for  Using a Bunsen Burner

As you know, the temperature of this burner is very high i.e.   1500 °C or 2700 °F, and therefore we have to follow safety rules to save from the occurrence of any incidents.

1. Always wear safety goggles and a lab apron when using this burner.
   If you have long hair, always tie it back.
2. Always light it with the air hole covered by the collar.
3. Always light a match or lighter and hold it above the Bunsen burner barrel before turning on the gas tap.
4. Never turn on a gas tap without a Bunsen attached and a match lit above the barrel.
5. Extinguish the match immediately after lighting it.
6. Always leave it on the yellow safety flame when you are not heating anything.
7. Always leave it on the blue heating flame when you are heating something.
8. Never put your hand in a flame.
9. Always extinguish a Bunsen flame by turning off the gas tap.
10. Never attempt to blow out a Bunsen flame.
11. If the flame accidentally goes out, turn the gas tap off immediately.
12. If there is a fire, immediately turn off the gas tap.

Key Notes on Bunsen Burner

1. It is one of the most requirements of a microbiology laboratory.
2. Using a Bunsen Burner
   Airhole fully open
   Type of flame: Roaring
   Purpose: To heat things fast
   Airhole half open
   Type of flame: Blue
   Purpose: To heat things slowly
   Airhole closed
   Type of flame: Safety flame
   Purpose: When we are not using the Bunsen but want to keep it on.
3. The mixture of air and gas (optimally about 1 part gas to 3 parts air)  forces by gas pressure to the top of the tube, where it ignites with a match.
4. A major purpose of the open flame in the aseptic technique is to create a cone of hot air above and around the laboratory bench to reduce the viability of organisms on suspended dust particles and thus creates a sterile zone for working.
5. The ability of its flame to heat things very quickly also makes it an ideal choice for sterilizing inoculating loops, warming glass bottlenecks, or igniting alcohol on culture spreaders, and so on.
6. Bunsen burner flames depend on airflow in the throat holes (on the burner side, note the needle valve for gas flow).

Incubator

Introduction of Incubator

The incubator is an electrical device in which the incubation process is performed which conditions an enclosure to a predetermined temperature since it provides and maintains all artificial optimal conditions for the growth of microbial culture as well as cell and tissue culture.

Principle and working of incubators

Incubators work on the principle of thermo-electricity. It has a thermostat that maintains a constant temperature by creating a thermal gradient. When any conductor, an electrically-controlled switch used for switching an electrical power circuit subjects to a thermal gradient, it generates voltage called as a thermoelectric effect. As power is supplied to the circuit predetermined temperature (37˚C) is set in the incubator. This temperature is maintained by the compatibility operation of the temperature sensor, temperature controller and temperature contactor are major components. When the power off, current flows into the system thereby energizing the contactor that powers the bulbs which serve as heating elements to the system, the fan ensures the Distribution of hot air in the entire system. When the temperature in the system gets to desirable i.e. 37˚C the digital temperature controller sends an electrical signal to the contactor which de-energized thereby switching off the heaters temporarily. Also when the temperature reduces beyond the desired temperature. The contractor will be energized again thus switch on the system.

Types of incubators

The most common types of incubators are –

1. BOD incubators
2. Bacteriological incubators
3. CO₂ incubators

The main difference between these two types of incubators is due to temperature. BOD stands for biological oxygen demand and it is the amount of dissolved oxygen needed by aerobic biological organisms to break down organic materials present in a given sample at a certain temperature over a specific time period.

Bacteriological incubators

This type of incubator is used mainly in laboratories usually for the growth of bacteria. A constant temperature set according to the requirement is possible because of having a thermostat that maintains it. Accurate temperature can be visible on the thermometer fixed on incubators. Most of the incubators are programmable which do not need trial and error temperature settings. They only hold a heating system that maintains the temperature for the growth of bacteria. Due to the absence of a cooling system, these incubators are affected by the temperature of the surrounding environment.

BOD incubators  (Low-Temperature Incubators)

These types of incubators are often called low-temperature incubators used for the growth of fungi i.e. yeast and mold as they require a low temperature to grow. These types of incubators are called BOD (Biological oxygen demand) incubators because in biological oxygen demand testing there is a need for a low temperature around 20-25˚C.  So, don’t confuse the term because the purpose of BOD incubatorS is also the same as bacteriological incubators.

CO₂ Incubators

Inside incubators, also known as a gassed incubators, an atmosphere is created that is as natural as possible to develop cell and tissue cultures. This way of cultivating living organisms is called in vitro and is the main application for CO₂  incubators.

The following parameters are crucial for cell cultivation:

• CO₂ level
• Temperature
• Humidity

Uses of incubators

Incubators use for the growth of microorganisms in many fields, including medical, pharmaceuticals, agricultural, environmental, food, and industrial microbiology, public health, basic research, and education. Whereas CO₂ incubators most frequently used in medical research and the pharmaceutical industry. However, they also provide sterile conditions for cultivation in other fields where cells must grow in a completely germ-free environment.

• CO₂ incubators for tissue-engineered products
• CO₂  incubators for in vitro fertilization
• CO₂  incubators in diagnostics
• CO₂  for developing biosensors
• CO₂  incubators in cancer research

Hot air oven

Introduction of Hot air oven

A hot air oven is the most common method of sterilization in the laboratory working on dry heat. Sterilization is the process of removing or destroying all microorganisms including viruses, bacteria, and their spores from the article or surface without destroying their quality and quantity. It is a physical method of sterilization due to dry heat. Factors influencing sterilization by heat are nature of heat i.e dry or moist, temperature and time, number of microorganisms, nature of microorganisms, type of microorganism, and presence of organic material. Mode of action: protein denaturation, oxidative destruction of essential cell constituents, and toxic effects of elevated levels of electrolytes. It works on the principle of conduction where heat is absorbed by the exterior surface of an item and then passed onward to the next layer. This method was introduced by Louis Pasture.

Principle of hot air oven

Electrical devices work on the principle of dry and hot air convection (that is circulation of heated air), conduction, and radiation. The hot air convection process is of two types. a. Gravity convection process: Heated air expands and possesses less density than cooled air which rises up and displaces the cooler air (the cooler air descends). It produces inconsistent temperature within the chamber thus has a slow turnover. b. Mechanical convection: Use of fitted blower or fan that actively forces heated air throughout all areas of the chamber. This dry heat destroys bacterial endotoxins (or pyrogens ) which are difficult to eliminate by other means. This property makes it applicable for sterilizing glass bottles that are to be filled aseptically. Dry heat kills by oxidation, protein denaturation, and toxic effects of elevated levels of electrolytes and it is more efficient.

Structure of Hot air oven and Functions

It consists of the following parts:

• An insulated chamber surrounded by an outer case containing electric heaters
• A fan
• Shelves
• Thermostat
• Door locking controls

Metallic cabinet with heating filament and fan fixed in the walls. Thermostat, temperature control, double-walled :(inner being a poor conductor and outer being metallic and air-filled space in between the layers) insulation keeps the heat in and conserves energy. Electrically heated, and provided with a fan or a blower to ensure rapid and uniform. Heating Mechanism:- Killing effect of dry heat on microorganisms is due to i) destructive oxidation of essential cell constituents, ii) protein denaturation and iii) toxic effect of elevated levels of electrolytes.

Uses of Hot air oven

Sterilization of articles that withstand high temperature and not get burned e.g. Glass-wares, powders,  forceps, scissors, scalpels, glass syringes,    pharmaceutical products like liquid paraffin, fats, grease and dusting powder, etc.

Handling procedure of Hot air oven

Wrap the articles or enclose them in a container of cardboard, aluminum, or paper. Mouths of flasks, test tube sand both ends of pipettes must be plugged with cotton wool. Articles to be sterilized such as Petri plates and pipettes may be arranged inside metal canisters and then placed. Place the articles at sufficient distances so as to allow free circulation of air in between them and to ensure uninterrupted airflow.  Shut the door and switch on the hot air oven. When the thermometer shows that the oven air has reached sterilizing temperature, heating is continued for the required period of time (e.g. 160°C for an hour). Allow the temperature to fall up to 40°C (approximately 2 hours), prior to removal of sterilized materials; which prevents breakage of glassware.

Advantages

1. Do not require water and there is not much pressure build-up within the oven making it safer to work.
2. Smaller than autoclave but can still be as effective.
3. Higher temperatures can be reached compared to other means.
4. This treatment kills the bacterial endotoxin, not all treatments can do this.
5. An effective method of sterilization of heat-stable articles only method of sterilizing oils and powders.
6. Protective of sharps or instruments with a cutting edge (fewer problems with dulling of cutting edges).
7. It does not leave any chemical residue.
8. It is non-toxic and does not harm the environment.

Disadvantage

1. Some organisms like prion may not be killed or inactivated.
2. Plastic wares or heat-sensitive materials can’t be sterilized.
3. Glasses may become smoky due to high sterilization temperatures: The temperature holding period is at 160°C for 1 hour, 170°C for 30 minutes whereas at 180°C for 20 minutes.
4. Dry heat penetrates materials slowly and unevenly and thus a time-consuming method because of the slow rate of heat penetration and microbial killing.
5. It requires a continuous source of electricity.

Sterilization control for hot air oven

A) Biological controls: 10⁶ spores of  Bacillus subtilis subsp. niger or spores of nontoxigenic strains of Clostridium tetani on paper strips are placed inside envelopes and then placed inside the hot air oven after complete sterilization inoculated in thioglycollate or cooked meat medium and incubated for sterility test under strictly anaerobic conditions for 3 to 5 days at 37°C. Growth in medium indicates the failure of sterilization.

B) Chemical control: Browne’s tube No. 3 shows a green color after sterilization at 160°C for 60 minutes ( color changes from red to green).

C) Physical control: Thermocouples and temperature chart recorder used.

Precautions

1. Sterilize dry substances.
2. It shouldn’t be overloaded.
3. Rubber goods, fabrics, any inflammable or volatile substances should not be put inside the oven.
4. The oven is allowed to cool gradually for about 2 hours or up to 40°C before the door is opened.

Autoclave

Introduction of Autoclave

An autoclave is the most common method of sterilization in the laboratory working on moist heat. Sterilization is the process of removing or destroying all microorganisms including viruses, bacteria, and their spores from the article or surface without destroying its quality and quantity. It is a physical method of sterilization due to moist heat. Factors influencing sterilization by heat are nature of heat i.e dry or moist, temperature and time, number of microorganisms, nature of microorganisms, type of microorganism, and presence of organic material. Mode of action, moist heat kills microorganisms by coagulating and denaturing their enzymes and their structural proteins. Heating in presence of water is preferred over dry heat because of its rapid killing and effectiveness even at a lower temperature than dry heat due to its latent heat. Temperature is above 100°C and sterilization by dry saturated steam under pressure. The most efficient method of sterilization is commonly called autoclaving and the instrument used is an autoclave.

Fundamental structures of Autoclave

Essentially a modified pressure cooker may be horizontal or vertical. It contains a double-walled or Jacketed chamber made of stainless steel or gunmetal with a supporting frame. In the modern type of autoclave, steam circulates within the jacket and is supplied under high pressure to the closed inner chamber where goods are kept for sterilization. One-fifth part of the cylinder is filled with water and the materials to be sterilized are placed inside. The lid is closed securely with a discharge tap on it open. A safety valve is present to permit the escape of steam from the chamber. It works on the principle of steam under pressure. It was invented by Charles Chamberland in 1879.

Principle of autoclaving

It utilizes the thermodynamics principle of water. Water boils when its vapor pressure equals that of the surrounding atmosphere. When the pressure inside the closed vessel increases, the temperature at which water boils also increases. Dry saturated steam at high pressure when strikes with the cooler surfaces of the articles in the autoclave, condenses into water, and efficiently destroys all microorganisms along with thermal resistant bacterial spores. Condensation of steam into water has 3 effects:

1. It wets the microorganisms and provides the essential conditions for killing.
2. Liberates latent heat of the steam and so rapidly heats up the items in the load. (Amount of heat liberated is 4 times greater than the heat available in the same mass of boiling water at the same temperature and pressure.
3. Causes significant contraction of steam, drawing more to the site. The cycle of condensation, the liberation of latent heat, and drawing of fresh steam is repeated until the article is heated up to the sterilizing temperature.

Types of autoclaves

According to structure

According to the structures, autoclaves are of following types and they are-
Simple non-jacketed autoclave, steam jacketed autoclave with automatic air and condensate discharge, and high pre-vacuum sterilizers.

According to function
According to function, autoclaves are of the following types and they are-Simple laboratory  autoclave, transportable benchtop autoclave, large simple autoclave, downward displacement laboratory  autoclaves, media preparatory autoclaves, and multi-purpose laboratory  autoclave

Sterilization cycle

The sterilization cycle includes warming of the chamber, vacuum extraction, pre-steam penetration time, steam penetration time, holding time, and cooling time.

Dynamics of sterilization
Thermal death time (TDT): It is the time in minutes required to kill all cells in a suspension at a given temperature. It is highly dependent on the inoculum size. Thermal death point(TDP):  It is the temperature needed to kill all cells in suspension after a fixed exposure time. D Value(decimal reduction time, DRT): It is the time in minute needed at a particular temperature to reduce the viable organisms by 90% i.e. to 10% or by 1 log 10. The value is independent of inoculum size and is inversely related to temperature.

Sterilization Times for Autoclave

Sterilization time is inversely proportional to the temperature at constant pressure. For examples  115°C, 10 lb/in 2 for 45 minutes; 121°C, 15 lb/in 2 for 15-20 minutes; 126°C, 20 lb/in 2 for 10 minutes and  134°C, 30 lb/in 2 for 3 minutes.

Sterilization Condition

Common sterilization condition is at 121°C for 15-20 minutes at 15 lb/sq inch but it may vary as-10 lbs pressure for 10 minutes is for culture media. 15 lbs pressure is for 20 minutes is for infected material. 20 lbs,30 minutes is for dressing and towels whereas 5 lbs-30 minutes is for gloves.

Sterilization Indicators

Automatic Process Control: It carries through the sterilization cycle according to a pre-selected scheme for the duration, temperature, and pressure of each stage. Recording Thermometer: Graphic record of temperature changes in chamber discharge channel avoiding errors in timing the holding period. Thermocouple: It is when kept inside the test article and attached to a potentiometer, it indicates the temperature inside the test article during autoclaving. Chemical Indicators: Browne’s Sterilizer has a red solution that turns green when heated at 115°C for 25 minutes (type 1), or 15 minutes (type 2). Store it at 20°C to avoid pre-mature color change. Adhesive Tapes: Bowie-Dick autoclave tape test for steam penetration. Biological indicators
Organism: Bacillus stearothermophilus (NCTC 10003 ATCC 7953), a thermophile that requires to be cultivated 55-60°C. Its spores are killed at 121°C in 12 minutes. Preparation: culture grown aerobically on nutrient agar for 5 days is suspended in sterile water to a concentration of one million spores per mili liter. Small strips of filter paper soaked in the suspension are dried at room temp and packed in envelopes.

Procedure of autoclaving

Initially check the water level. Place the articles to be sterilized in the center of the largest or most densely packed items and some in the coolest part. Now switch on autoclave. Check the pressure and wait for the proper time (If there is an automated system, no need for these steps). After autoclaving, the envelope is cut with sterile scissors and strip transferred to a recovery medium, e.g. thioglycolate broth with strict precautions against contamination. Incubate tube for 7 days at 55°C and examined for growth. An unautoclaved spore strip is used as positive control and an uninoculated tube of the medium as a negative control. Use results in terms of the degree of heat resistance of preparation.

Uses of autoclave

It is widely used for the following purposes and are-sterilization of culture media, aqueous solutions, empty bottles, and impervious containers, surgical instruments, wrapped dry goods and dressings, gowns and dressings, rubber goods, syringes, etc.
Advantages:  It is a very effective way of sterilization, quicker than a hot air oven.
Disadvantage: Articles may trapped air, takes a long time to cool.

Precautions

Air must be completely removed until saturated steam is filled. Contents should be arranged loosely to ensure free circulation of steam. It shouldn’t allow forming supersaturated steam. The lid should be opened only after pressure gets down to normal. Follow manufacturers’ guidelines. Avoid standing directly in front of the autoclave door when opening. Cool to below 80°C before opening.

Mechanism of Bacterial Spores Resistance

There is no clear and proved concept but several theories are as follows-Spores have low water content and therefore essential factor in resistance.
-According to Warth (1985), the stability of protein could be intrinsically, or due to the presence of a substance ( might be Calcium-diplocolinate) that helps to stabilize or due to the removal of water. Several properties of spore i.e. thermotolerance, mineralization, thermal adaptation might help in resistance. Small acid-soluble spore proteins(SASPs) may also play some role.

Microscope

Introduction of Microscope 

• A microscope is an instrument, the most characteristic of microbiology laboratories. The magnification, it provides, enables us to see microorganisms and their structures otherwise invisible to the naked eye. The magnitudes attainable by microscopes range from 100 X-10,000,000X. It may be defined as an optical instrument, consisting of a lens or a combination of lenses, for making enlarged or magnified images of minute objects. (Micro: Small; scope: to view)
• Antony Von Leuwenhoek is considered to be the first person who has seen a microorganism through a simple microscope made by him with a magnification of 270-480 times.
• He described the size, shape, movement of bacteria, protozoa, and algae. These findings were confirmed after the development of a compound microscope by Robert Hooke.
• The characteristic morphological studies enabled by the discovery of powerful microscopes, helped scientists to classify microorganisms. Later improvements in the compound microscopes were made and Amici discovered an oil immersion lens, which enabled the scientists to study the characteristics more minutely. Microscopes are continuously improved to enable us to have higher magnifications and better resolutions.
• The light microscope, in which the magnification is obtained by a system of optical lenses uses light waves. Whereas the electron microscope uses a beam of electrons in place of light waves for visualization of objects.
• Compound Microscope: It is most commonly used in microbiology laboratories. In compound microscopy, the microscopic field or area observed is brightly lighted and the objects being studied appear dark because they absorb some of the light. Ordinarily, microorganisms do not absorb much light but staining them with a dye greatly increases their light-absorbing ability resulting in greater contrast and color differentiation.
• Generally, microscopes of this type produce a useful magnification of about 1000X to 2000X.

Simple Microscopes

Principle of Simple/ Dissecting Microscopes:

The dissecting microscope consists of a biconvex lens which is moved up and down by an adjustment screw to bring the object in sharp focus. The object is placed on the platform and light is focused with the help of a concave mirror fitted below

In simple microscopes, a convex lens of short focal length is used to see magnified images of a small object. The object is placed between the optical Centre and the focus of a convex lens, its image is virtual, erect and magnified, and on the same side as the object. The position of the object is so adjusted that the image is formed at the least distance of distinct vision.

Principle of Compound Microscopes

• A compound microscope consists of two sets of convex lenses. A lens of short aperture and short focal length facing the object is called objective. Another set of the lens of relatively moderate focal length and large aperture facing the eye is called the eyepiece. The objective and the eyepiece are placed coaxially at the two ends of a tube.
• The object is placed between the centre of curvature and focus of the objective – it forms a real, inverted, and magnified image on the other side of the objective. This image acts as an object for the eyepiece which then acts as a simple microscope to produce virtual, erect, and magnified images.

Parts of Compound Microscope

• It consists of

1. Microscope stand
2. Stage
3. Microscope Optics

Microscope stand

It is the main framework of the microscope and consists-

• Main tube
• Body tube
• An arm, which supports the main tube, body, and the stage
• A substage, and
• A foot or base upon which the whole instrument rests.

Main tube: The main tube primarily holds the objective and eyepieces. The eyepiece, also known as an ocular piece, is present at the top of the main tube.

Body and arm: The tube is attached to the microscope by the component of the microscope called the body. The body of the microscope and the tube attached to it are supported at the correct height by a firm arm, which may also provide a lifting handle for the microscope.

Substage: The sub-stage lies immediately below the stage. This holds a condenser lens with an inbuilt diaphragm and a holder for light filter and stops.

Foot: The microscope rests firmly on the laboratory bench with the base called a foot. This may be U-shaped or rectangular.

Stage

A fixed platform with an opening in the Centre allows for the passage of the light from an illuminating source below to the lens system. It provides a surface for the placement of a slide over the central opening.

Microscopic optics

These include

• objective
• eyepieces
• illuminating source

Mechanical Adjustment of a microscope:

It is being carried out to focus the specimen examined by the microscope. This adjustment includes coarse and fine focusing adjustments and condenser.

Coarse adjustment- required when focusing the specimen with low power (10X) objectives.

Fine adjustment-It is carried out when finer focusing is required by using high power (40X) objectives or oil immersion objectives.

Condenser adjustment-It is classified depending on its uses such as bright field, darkfield, phase contrast, etc.

The light source: A good source of light is needed to examine the specimen correctly. This may be daylight or electric light.

Magnification

The purposes of microscopes are to produce an enlarged, well-defined image of objects too small to be observed with the naked eye.

Magnification: Objective lens× eyepiece

Three objectives most commonly used are

-10X, 40X and 100X

and eyepieces use 5X, 10X and 15X

Terminology

Numerical Aperture: It may be defined as the ratio of the diameter of the lens to its focal length.

Resolution: Resolving power is the ability to reveal two closely adjacent structural details as separate and distinct.

The greatest resolution in the light microscope is obtained with a shortest wavelength of visible light and object with maximum numerical aperture.

Illumination: Effective illumination is required for efficient magnification and resolving power. Since the intensity of the daylight is an uncontrolled variable, artificial light from a tungsten lamp is the most commonly used light source in microscopy.

Difference between simple and compound microscopes

Sizes of Microorganism

Bacteria

Cocci 0.5-1.0 µm while bacilli 1-10 µm×3-10µm

Viruses

Smallest-Parvo virus-20 nm while largest –Pox virus-300 nm

Parasites:

Most protozoa around 50µm in size except Balantidium coli ≥100µm

Helminthes sizes are variable-

Cestodes 1 mm to several meters in length

Nematodes vary in size from  5 mm to even 1 meter

Fungus

Yeast like appearance 2-30µm

Mould appearance 2-5 µm

Spherical like appearance 5-300µm

Handle and  care  of microscopes

Handle with care

Most microscope problems occur as a result of improper handling. When carrying it, hold it by the base and the metal support arm. Do not pick it up by the stage, as this can cause misalignment. When transporting it, use its own bag.

Examination of the slide should always begin with a low power objective (10X).

Keep lenses clear of slides

When using microscopes and adjusting the focus you will need to lower the objective lens down as far as it will go. However, you should never allow the lens to touch the slide you are looking at. Dirty lenses can be difficult to clean.

Clean after using immersion oil

If using immersion oil, always ensure the objectives are cleaned immediately after use. Objective, eyepieces, and condenser may be removed for cleaning. Use only lens paper and lens cleaner. Do not use solvents.

Cover when not in use

All microscopes are sold with dust covers. Always keep it covered when not in use even if it is stored in a cabinet. Eye tubes also need to be kept free of dust so do not store a microscope without the eyepieces. If the microscope eyepieces must be removed, cover the tubes with caps or a plastic bag with a rubber band around the eye tube.

Look after the bulb

After using the microscope, turn off the illuminator and wait for it to cool for several minutes before putting it away. By allowing the bulb to cool you will extend its life. When turning the microscope on and off, use the switch, not the PowerPoint. Do not switch it on while using full light intensity. Never touch the bulb with your fingers as the body oils can burn into the bulb and reduce its life. Use a tissue. Keep a store of replacement bulbs and always use the correct bulb.

 Store in a clean, dry place and away from direct sunlight

Make sure you do not store your microscope in an area that has corrosive chemical fumes that can destroy lenses or metal parts or beside solutions that may leak. Salt air and pervasive damp can also cause damage over time. Make sure your cabinet is ventilated.

Only use special lens paper or wipes for cleaning the lenses

Lenses can easily be scratched and should be treated with great care. Use an aspirator to remove dust. Sticky residue can be removed with lens paper moistened with distilled water or lens cleaning solution and rubbed gently using a circular motion. Never use sharp instruments or anything abrasive on the microscope lenses.

Keep your User’s Manual and wrenches in a safe place

Each microscope should come with a user’s manual and specialist wrenches as required. Always consult the User’s Manual before making any adjustments to your microscope and use the wrenches provided. Never over-tighten or use force when performing any maintenance on your microscope, or use inappropriate tools. This can damage the parts.

An attempt should never be made to repair the microscope by oneself.

Key Notes

On the basis of the lens, the microscope is of two types, simple and compound microscope.

Magnification based principle, the microscope is mainly two types-

• Light or Optical Microscope  e.g.

√Bright Field

√Dark Field

√Fluorescence  and

√Phase Contrast Microscope

• Electron Microscope e.g.

√Transmission and

√Scanning

Preparation of different media in Pharmaceutical  Microbiology 

Introduction of culture media

Media is plural while medium singular. Culture media require to grow the organisms from infected material to identify the causative agent. They are of different types on the basis of using purposes. Nutrient agar(NA) uses for the cultivation of non-fastidious bacteria like Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa whereas 5% of sheep blood agar (BAP) needs for Streptococcus species, Neisseria species. Chocolate agar(CHOC) is useful for the culture of Haemophilus influenzae. MacConkey agar (MAC) is a selective, differential, and indicator medium and uses for the cultivation of Gram-negative bacteria. All the organisms growing on nutrient agar, blood agar, MacConkey agar can easily grow on chocolate agar but not vice -versa.

Composition of culture media

Water is the source of hydrogen and oxygen. As electrolyte Sodium chloride or other electrolytes are necessary. Peptone is a complex mixture of partially digested proteins. It contains proteoses, amino acids, polypeptides, phosphates, minerals (K, Mg), and accessory growth factors like nicotinic acid and riboflavin. Meat extract is available commercially as “Lab-Lamco”. It contains protein degradation products, inorganic salts, carbohydrates, and growth factors. Blood or serum uses for enriching culture media. Usually, 5-10% defibrinated sheep blood uses. In certain media, serum uses. Agar is a long-chain polysaccharide and prepared from sea wood (Algae –Geladium Species). It does not provide any nutrition to the bacteria but acts as a solidifying agent only. It uses in the concentration of 2-3%. It melts at 98°C and solidifies at 42°C. New Zealand agar has twice the jellifying capacity as that of Japanese agar.

Types of  culture media

Media are of flowing types.

Based on the physical state

I. Liquid media

II. Semisolid media ( Agar, 0.2-0.4% which enables motile bacteria to spread.)

III. Solid  media

On the basis of the presence of molecular oxygen and reducing substances in the media

I . Aerobic media and

II. Anaerobic media

Based on  nutritional factors

I. Simple media

II. Complex media

III. Synthetic media and

IV. Special media

Special culture media –

a )Enriched media

b) Enrichment media

c) Selective media

d) Differential media

e) Indicator media

f)Transport media and

g) Sugar media

Simple media

The nutrient broth is an example of a simple medium. It contains peptone water and meat extract 1%. when agar adds to nutrient broth, it becomes nutrient agar. This is the simplest and routinely employed medium in the laboratory for diagnostic purposes.

Complex media

All media other than simple media are complex media.

Synthetic media:

They prepare from pure chemicals and the exact composition of the medium is known. These uses for special studies such as metabolic requirements. Dubbo’s medium with tween 80 is an example of a synthetic medium.

Special Media

Enriched media: When a basal medium is with the addition of some nutrients such as blood, serum, or egg. It becomes an enriched medium. For e.g. in blood agar-there is the addition of blood to nutrient agar. It may use for growing a number of bacteria but one specific example is Streptococcus that requires blood for its growth. In Loeffler’s serum slope, there is the addition of serum for enriching the medium. This medium uses for growing Corynebacterium diphtheriae.

Enrichment media

A fluid type of selective medium in which some substances incorporate that have either a stimulating effect on the bacteria to be grown or inhibits its competitors or both. This results in an absolute increase in the number of wanted bacteria related to other bacteria. Such medium is enrichment medium.  The addition of tetrathionate in tetrathionate broth inhibits coliforms while allows typhoid-paratyphoid bacilli to grow.  In selenite F broth, selenite has a similar action as that of tetrathionate in tetrathionate broth.

Selective media

Selective media contain substances that inhibit all but a few types of bacteria and facilitate the isolation of a particular species. These media use to isolate a particular bacteria from a specimen where mixed bacterial flora has expected. Selective media are solid in contrast to enrichment media which are liquid. Examples of selective media are deoxycholate citrate agar(DCA)-The addition of deoxycholate acts as a selective agent for enteric bacilli (Salmonella, Shigella) and bile salt agar(BSA). Bile salt is a selective agent. It favors the growth of only Vibrio cholerae whereas inhibits the growth of other intestinal organisms.

Differential  media

When a medium contains substances that help to distinguish differing characteristics of bacteria, is a differential medium e.g. MacConkey’s medium, which contains peptone, lactose, agar, sodium taurocholate, and neutral red. The lactose fermenters (LF) form pink-colored colonies whereas non-lactose fermenters (NLF) produce colorless or pale colonies.

Indicator Media 

These media contain an indicator that changes color when a bacterium grows in them. Salmonella enterica serotype Typhi grow as black colonies on Wilson and Blair’s medium containing sulfite. MacConkey ‘s medium is also an indicator medium. Due to the fermentation of lactose, there is acidic pH which forms the pink colonies in the presence of a neutral red indicator.

Transport media

They use in the case of delicate organisms (e.g. gonococci) which may not survive the time taken for transit or may be overgrown by non-pathogenic bacteria (e.g. cholera organisms). They maintain only viability. Examples of transport media are Stuart’s transport medium: is a non-nutrient soft agar gel containing a reducing agent to prevent oxidation, and charcoal to neutralize bacterial inhibitors. It may be for organisms such as gonococci. Buffered glycerol saline transport medium for enteric bacilli.

Sugar media

Sugar media help in the identification of bacteria. The term sugar in microbiology denotes any fermentable substance. Glucose, lactose, sucrose, and mannitol routinely employ for fermentation tests.

Anaerobic Media

These use for the cultivation of anaerobic bacteria e.g. Robertson’s Cooked meat medium and thioglycollate broth.

Blood  agar preparation

Introduction of Blood Agar

Blood agar is an enriched medium for bacteria. Fastidious organisms, such as streptococci, do not grow well on ordinary growth media. It is a type of growth medium i.e. trypticase soy agar enriched with 5% sheep blood or blood agar base with 5-10 % sterile sheep blood that encourages the growth of bacteria and their hemolysis, such as streptococci, that otherwise wouldn’t grow.

Composition of Sheep Blood Agar Base

Ingredients            Gms / Litre

Casein enzymic hydrolysate   14.000

Peptic digest of animal tissue   4.500

Yeast extract  4.500

Sodium chloride  5.000

Agar  12.500

Final pH (at 25°C)  7.3±0.2

Procedure for the Preparation of Blood Agar

1. Suspend 40.5 grams in 1000 ml distilled water or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Cool to 45-50°C and aseptically add 7% sterile sheep blood.
5. Mix well and pour into sterile Petri plates. Avoid the formation of air bubbles. You must have warmed the blood to room temperature at the time of dispensing to a molten agar base.
6. Dispense 15 ml amounts to sterile Petri plates aseptically
7.  Label the medium with the date of preparation and give it a batch number (if necessary).

Storage and Shelf Life

Store below 30°C in a tightly closed container and the prepared medium at 2-8°C, preferably in sealed plastic bags to prevent loss of moisture. The shelf life of thus prepared blood agar is up to four weeks. Use before the expiry date on the label.

Principle and Interpretation

Hemolysins are exotoxins produced by bacteria that lyse red blood cells. The hemolytic reaction can be visualized on blood agar plates observing through the bright transmitted light. On blood agar plates colonies of hemolytic bacteria may be surrounded by a clear, colorless zone where the red blood cells have been lysed and the hemoglobin destroyed to a colorless compound and which is beta hemolysis. Other types of bacteria can reduce hemoglobin to methemoglobin which produces a greenish zone around the colonies and is called alpha hemolysis. Gamma hemolysis is lacking hemolysis where no change in the medium is observed. Sheep blood agar base with added sheep blood was developed to allow maximum recovery of organisms without interfering with their hemolytic reactions. Sheep blood agar base was formulated to be compatible with sheep blood and give improved hemolytic reactions of organisms. Casein enzymic hydrolysate and yeast extract provide nitrogen, carbon, amino acids, and vitamins. Peptic digest of animal tissue (PDAT) is the nitrogen source. Sodium chloride (NaCl) maintains the osmotic balance. Sheep blood agar base showed considerable improvement and the expected beta-hemolytic reactions with S. pyogenes in comparison to other blood agar bases supplemented with blood

Quality control

• Inoculate Streptococcus pneumoniae and Streptococcus pyogenes into a prepared blood agar.
• Observation of Cultural characteristics after incubation at 35-37°C for 18-24 hours with added 7% v/v sterile sheep blood.

Organism                                              Growth                             Haemolysis

• Streptococcus pneumoniae

ATCC 6303                                       luxuriant                            alpha

• Streptococcus pyogenes

ATCC 19615                                          luxuriant                      beta

• This is an indicator of good quality control of prepared blood agar.

Uses of Blood Agar

Blood agar has the following uses:

1. Determine the type of hemolysis, whether alpha, beta, or gamma.
2. For the culture of streptococci as well as antimicrobial susceptibility testing (AST).
3.  Use of optochin disc for presumptive identification of  Streptococcus pneumoniae.
4. Similarly use of bacitracin disc (0.04U) for presumptive identification of Streptococcus pyogenes.
5.  To perform CAMP test for Streptococcus agalactiae.
6. To perform satellitism test for Haemophilus.
7. It is also used for the isolation and cultivation of other than streptococci like Neisseria and other fastidious microorganisms.
8. It is also useful for the preparation of Salmonella Typhi antigens.

Keynotes

1. Blood is a good constituent of enriched medium for fastidious organisms even though contains inhibitors for certain bacteria such as Neisseria and  Haemophilus genera and the blood agar must be heated to inactivate these inhibitors and to release essential growth factors (e.g., V factor). The heating of blood agar converts it into chocolate agar (75°C for 15 minutes turns a chocolate color) and supports the growth of these bacteria.
2. Hemolysis on blood agar: Mainly three types of hemolysis are produced in Sheep blood agar by Streptococci namely; Alpha hemolysis, Beta hemolysis, gamma hemolysis but sometimes alpha prime or wide zone alpha hemolysis may be encountered. Hemolysis is best observed by examining colonies grown under anaerobic conditions or inspecting sub-surface colonies. How does one know if the colonies they are observing on a plate have caused alpha hemolysis or beta hemolysis or gamma ?-Follow up principle and interpretation.
3. To check the type of blood agar hemolysis, the blood agar plate must be held up to a bright transmitted light source and observed with the light coming from behind.
4. Alpha hemolysis: Streptococcus pneumoniae
5. Beta Hemolysis: Group A beta-hemolytic streptococci-Streptococcus pyogenes and Group B, beta-hemolytic streptococci–Streptococcus agalactiace. For the group, A  streptococci maximal activity of both the hemolysins; Oxygen labile SLO, and oxygen stable SLS hemolysins is observed only in anaerobic conditions.
6. Gamma or non-hemolysis: Enterococcus species
7. Alpha prime or wide zone alpha hemolysis: A small zone of intact erythrocytes immediately adjacent to bacterial colony, with a zone of complete red-cell hemolysis surrounding the zone of intact erythrocytes. This type of hemolysis may be confused with Beta hemolysis.
8. The double zone of hemolysis on it also seen by some organisms like Clostridium perfringens and Aeromonas hydrophilia called target hemolysis.
9. If you are planning to prepare a batch of blood agar plates, prepare few blood agar plates first to ensure that blood is sterile.
10. CAMP test for Streptococcus agalactiae. -positive on blood agar as shown below-

S. penumoniae antimicrobial susceptibility testing (AST) on blood agar as shown below-

S. penumoniae optochin test on blood agar as shown below-

Growth of Staphylococcus (pinhead colony) and Streptococcus (pinpoint colony) both are beta-hemolytic  on blood agar as shown below-

Growth of Streptococcus pyogenes on blood agar, beta-hemolytic colonies, and 0.04 Unit bacitracin sensitive as shown below-

Staphylococcus and Enterococcus on blood agar and use of catalase test as shown below-

Viridans Streptococci growth on blood agar and optochin resistant as shown below-

Nutrient agar and both

Introduction of Nutrient agar and Broth

Nutrient agar is the simple medium which uses to grow bacteria. It is devoid of indicator, selective agent, differential ingredients and enriching substances therefore use for better expression of pigmentation, biochemical test, and even for serotyping. The nutrient broth is the liquid medium and the composition is the same as the nutrient agar except for agar.

Composition of Nutrient Agar

(Oxoid)
Typical Formula                      gm/litre`

Lab-Lemco’ powder:                 1.0
Yeast extract:                              2.0
Peptone:                                         5.0
Sodium chloride:                       5.0
Agar:                                               15.0

Distilled water:                        1000 ml

pH 7.4 ± 0.2 @ 25°C

Principle of Nutrient Agar

Peptone is an enzymatic digest of animal protein and the principal source of organic nitrogen for the growing bacteria. Lab-Lemco powder( beef extract) and yeast extract are water-soluble ingredients of nutrient agar which contribute vitamins, carbohydrates, nitrogen, and salts. Sodium chloride maintains the osmotic equilibrium of the medium. The presence of sodium chloride in nutrient agar maintains a salt concentration in the medium that is similar to the cytoplasm of the microorganisms. Agar acts as the solidifying agent.  Water is an essential ingredient for the growth and reproduction of organisms and also serves as a transport medium for the agar’s various substances.

Preparation of Nutrient agar

1. Suspend 28.0 grams in 1 liter purified/distilled or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. After autoclaving,  leave for cooling to 45-50°C.
5. Pour nutrient agar into each plate and leave plates on the sterile surface until the agar has solidified.
6. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Nutrient agar

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
• The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

• Test specimens ( samples or growth of bacteria)
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (Escherichia coli ATCC 25922 and Staphylococcus aureus
   ATCC 25923)

Test procedure ( specimen/organism inoculation)

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Inoculate and streak the specimen as soon as possible after collection.
3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
4. Streak for isolation with a sterile loop.
5. Incubate plates aerobically at 35-37ºC. for 18-24 hours.
6. Examine colonial characteristics.

Result Interpretation

Control strains i.e. Escherichia coli ATCC 25922 and Staphylococcus aureus
ATCC 25923): good-luxuriant

Presence of non-fastidious bacteria in specimen: Presence of  growth on nutrient agar

Colony Characteristics of various organisms in Nutrient Agar

Colony morphology of various bacteria as shown in following video-

Colony of Staphylococcus aureus on nutrient agar golden yellow large i.e. greater than 1 mm in size, smooth, convex, opaque, and easily emulsifiable

Pyocyanin and pyoverdin din pigments of Pseudomonas on nutrient agar: Pigment production of Pseudomonas, some strains produce diffusible pigments like Pyocyanin (blue), pyoverdin or fluorescein ( greenish-yellow), pyorubin (reddish-brown), and pyomelanin ( brown to black). There are 6 different types of Pseudomonas aeruginosa colonies that may be observed- Type 1. Large and leafy 2.  smooth, circular, and coliform type Type 3. Rough Type 4.  rogose Type 5. Mucoid due to exopolysaccharide as shown above image and Type 6.  Dwarf and smallest

Colony morphology of bacteria || Micrococcus roseus || Red pigment on nutrient agar as shown below-

Uses of Nutrients Agar

1. For the cultivation and maintenance of non-fastidious bacteria.
2. Preparation of blood agar
3. It is also used antibiotic sensitivity testing
4. Concentrated agar up to 3 more % prevents swarming of Proteus species as well as Clostridium tetani.
5. Preparation of chocolate agar ( heating blood agar changes to chocolate agar).
6. It uses for better expression of pigmentation.
7. It is also used for serotyping of organisms.
8. It also uses for the isolation of pure cultures from mixed growth.
9. Nutrient agar is also beneficial for the enumeration of organisms in water, sewage, dairy products, feces, and other materials.

Key Notes

• Nutrient broth contains the same ingredients except for agar.
• Earlier it was used as blood and chocolate agar base medium but nowadays replaced various manufacturers.

Limitations of Nutrient Agar

1. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
2. Each lot of the medium has been tested for the organisms specified on the certificate of analysis. It is recommended to users to validate the medium for any specific microorganism other than mentioned in the certificate of analysis (COA) based on the user’s unique requirement.
3. It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
4. It is a general-purpose medium and thus for recovery of fastidious organisms like S. pneumoniae and H. influenzae nutrient agar should be modified.

Soybean casein digest broth

…

Sabouraud dextrose agar

Introduction of SDA

SDA stands for Sabouraud Dextrose Agar and  Sabouraud is the surname of creater Raymond Jacques Adrien Sabouraud a French physician born in Nantes. He formulated SDA in 1892 for culturing dermatophytes. SDA  is the most common fungal medium to recover the growth of fungi in Mycology. It is different from bacterial medium due to having main two properties, comparatively low pH (5.6) and high concentration of sugar. It is useful for the isolation, cultivation, and maintenance of non-pathogenic and pathogenic species of fungi ( yeasts and molds). The pH is adjusted to approximately 5.6 in order to enhance the growth of fungi, especially dermatophytes, and to slightly inhibit bacterial growth in clinical specimens.

Principle of SDA 

Carlier’s modification of SDA contains ingredients like dextrose, mycological, peptone, agar, and pH 5.6. Mycological peptone provides nitrogen and vitamin source required for organisms in this medium. Dextrose provides an energy source while agar acts as a solidifying agent. High dextrose concentration and low pH favor fungal growth and inhibit contaminating bacteria from test specimens. This modification is useful for the cultivation of fungi (yeasts, molds), particularly useful for the fungi associated with skin infections. This medium is also employed to determine microbial contamination in food, cosmetics, and clinical samples.

Composition of SDA

Sabouraud  original formulation

• Peptone:10 g
• Glucose: 40 g
• Agar: 15 g
• Distilled Water (D/W): 1000 ml

Note:  The acidic pH of traditional Sabouraud agar inhibits bacterial growth.

Emmon’s modification

• Neo-peptone: 10 g
• Glucose: 20 g
• Agar: 20 g
• Distilled Water (D/W): 1000 ml

Note: Shifting pH towards neutral and lowered concentration of sugar of the Emmon’s modification enhances to support the growth of other microorganisms and some pathogenic fungi also, such as dermatophytes.

Carlier’s modification

Ingredients                         Gms / Litre
Dextrose (Glucose) :       40.000
Mycological, peptone:  10.000
Agar:                                       15.000
Final pH ( at 25°C:          5.6±0.2

Note: High sugar (dextrose) concentration and low pH(5.6) favor fungal growth and inhibit contaminating bacteria from test specimens.

Modified SDA with the incorporation of antimicrobial agents

(Gentamicin, chloramphenicol, tetracycline, cycloheximide)

1. SDA with chloramphenicol contains 50.0 mg of chloramphenicol and the final pH of the medium should be 5.6 +/- 0.3 at 25ºC. Chloramphenicol inhibits a wide range of gram-positive negative bacteria.
2. SDA with chloramphenicol and gentamicin contains 50.0 mg of chloramphenicol and 5.0 mg gentamicin. The final pH of the medium should be  5.6 +/- 0.3 at 25ºC. Chloramphenicol inhibits a wide range of gram-positive negative bacteria whereas gentamicin inhibits gram-negative bacteria.
3. SDA with chloramphenicol and tetracycline contains 50.0 mg of chloramphenicol and 10.0 mg of tetracycline. The final pH of the medium should be 5.6 +/- 0.3 at 25ºC. Chloramphenicol inhibits a wide range of both gram-positive negative bacteria while tetracycline inhibits a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites.
4. SDA with cycloheximide: Cycloheximide inhibits primarily saprophytic fungi but not dermatophytes or yeasts. It is incorporated as an amount of 0.5 g/liter.

Note: Antimicrobial agents should only be added after media has been autoclaved and then cooled to 45 to 50°C. Keep all plates at 4°C until they are used, regardless of whether they contain antibiotics.

Preparation of SDA various modifications

SDA original (Sabouraud formulation)

1. Add all ingredients in nearly 900 ml of distilled water or deionized water.
2. Adjust pH to 5.6 with hydrochloric acid.
3. Then adjust the final volume to 1 liter.
4. Autoclave 20 minutes at 121°C, 15 lbs.
5.  Cool to 45 to 50°C and pour into Petri plates or tubes for slants.

Note: The addition of antimicrobial agent, chloramphenicol is optional and its use depends on the user’s preference. If desired add 5 mg to the above recipe.

Emmons modification SDA

• Add all ingredients in nearly 900 ml of distilled water.
• Adjust pH to 6.8 to 7.0 with hydrochloric acid (HCl).
• Then adjust the final volume to 1 liter.
• Sterilize by autoclaving 20 minutes at 121°C, 15 lbs.
•  Cool to 45 to 50°C and pour into Petri plates or tubes for slants.

Carlier’s modification of SDA

1. Suspend 65.0 grams in 1000 ml distilled water.
2. Heat to boiling to dissolve the medium completely.
3. Autoclave at 15 lbs pressure (121°C) for 15 minutes.
4. Cool to 45-50°C.
5. Mix well and pour into sterile Petri plates or test tubes.
6. Store prepared SDA plates or tubes at 2-8°C until any defects appear on them.

Requirements for testing SDA

• SDA
• Suspected fungal specimen
• Quality control strains ( as positive controls: Positive controls:
  Candida albicans ATCC® 10231 and Aspergillus brasiliensis ATCC® 16404 while as negative control uninoculated medium)
• Incubator/s
• Biological safety cabinet (BSC)
• Inoculating loop  or wire
• Bunsen burner
• Personal protective equipment (PPE) as required

Test procedure

1. Inoculate the specimens on SDA.
2. Incubate the medium ( according to the required temperature and time depending on the nature of fungi to be recovered e.g. as you know, dimorphic fungi occur in two forms-yeast forms ( parasitic phase) and mold ( spores and filamentous form). Typically, molds are incubated at room temperature (22 to 25°C) and yeasts are incubated at 37°C if suspected of being dimorphic fungi. Incubation times will vary, from approximately 2 days for the growth of yeast colonies such as Malasezzia species, to 2 to 4 weeks for growth of dermatophytes ( Trichophyton, Microsporum, and Epidermophyton) or dimorphic fungi such as Histoplasma capsulatum. Indeed, the incubation time required to acquire fungal growth is one of the  diagnostic tools used to identify or confirm fungal species.)
3. Observe for fungal growth.

Result Interpretation of fungal growth on Sabouraud Dextrose Agar

• Yeasts: Creamy to white colonies
• Molds: Filamentous colonies of various color
• Positive controls: Candida albicans: Luxuriant
  (white colonies)
• Aspergillus brasiliensis : White mycelium; black spores
• Negative control:  No change in Uninoculated medium( SDA)

Uses of SDA

1. SDA is recommended for the cultivation of yeasts, molds, and aciduric bacteria from clinical samples.
2. The medium with the addition of antimicrobial agents (antibiotics) makes it more selective for the isolation of pathogenic fungi from material containing large numbers of microbial load i.e. other fungi or bacteria.
3. This medium is also applicable to determine microbial contamination in food and cosmetics specimens.

Limitations of SDA

1. For heavily contaminated specimens, the plate must be supplemented with inhibitory agents for inhibiting bacterial growth with lower pH.
2. Avoid overheating SDA medium during preparation with an acidic pH, this may result in a soft medium
3. SDA is not recommended as a primary isolation medium because it is insufficiently rich to recover certain fastidious pathogenic species, particularly most of the dimorphic fungi like e.g. Blastomyces dermatitidis, coccidioides immitis, Histoplasma capsulatum.
4.  Some pathogenic fungi may produce infective spores which are easily dispersed in the air, so examination should be carried out in a safety cabinet.
5. It does not promote the conidiation of filamentous fungi.
6. Further biochemical or serological tests should be performed for confirmation
7. For identification, organisms must be in pure culture.
8. Antibiotics added into a medium to inhibit bacteria may also inhibit certain pathogenic fungi.

Keynotes of Sabouraud Dextrose Agar

• Colony characteristics,  microscopic structures, rate of growth, media which support the growth of the organism, and source of the specimen are very helpful for the isolation of fungi.
• A variety of biochemical tests are available for the identification of yeasts.
• Sabouraud Dextrose Broth (SDB) is the same formulation as SDA, without agar.

Colony Characteristics of various fungi ( yeasts and molds) are given below-

Sporothrix schenckii growth on SDA

Acremonium on SDA and LPCB preparation

Aspergillus fumigatus Colony on SDA, LPCB tease mount under microscopy

Identification of Microorganism by different staining techniques (Gram’s staining, LPCB mount) in Pharmaceutical  Microbiology 

Introduction of Staining

Staining is a method used to enhance contrast in specimens,  normally at the microscopic level. Stains and dyes are frequently used in histology and in the medical fields of microbiology to stain microorganisms like bacteria, fungus, parasites, etc., histopathology, hematology, and cytopathology that focus on the study and diagnoses disease at a microscopic level. Stains may be used to define biological tissues, cell populations e.g. blood cells, or cell organelles within individual cells. Similarly in biochemistry, it involves adding a class-specific like DNA, proteins, lipids, carbohydrates dye to a substrate to qualify or quantify the presence of a specific compound. Staining and fluorescent tagging are also useful that can serve similar purposes. Biological staining is also used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel electrophoresis but here we concern with the field of medical or clinical microbiology.

❖Stain: to leave a mark on something that is difficult to remove
❖Dye: A dye is a colored substance that has an affinity
to the substrate to which it is being applied.

❖Staining: Staining is an auxiliary technique used in
microscopy to enhance contrast in the microscopic
image

Types of  staining 

1. Simple Staining
2. Gram’s Staining
3. Acid Fast Staining
4. Albert’s Staining
5. Capsule Staining
6. Spore Staining
7. Negative Staining

Simple Staining

Simple staining employs staining of bacterial smear with a single staining reagent. The commonly used simple stains are the basic stains such as methylene blue, crystal violet, and carbol fuchsin. They provide good color contrast and impart the same color to the stained organisms. Example of simple staining -WBCs stained by New methylene blue as shown below

Pus cells in supravital stain ( New methylene blue)

Pus cells, RBCs, and C L crystals in the pleural fluid under the microscope ( without any stain)

Gram’s Staining

It is a differential stain and thus used to differentiate Gram-positive and Gram-negative bacteria. It was originally devised by a Danish bacteriologist, Hans Christian Joachim Gram (1884) as a method of staining bacteria.

Principle of Gram stain

The reaction is dependent on the permeability of the bacterial cell wall and cytoplasmic membrane, to the dye–iodine complex. In Gram-positive bacteria, the crystal violet dye iodine complex combines to form a larger molecule which precipitates within the cell. Also, the alcohol /acetone mixture which acts as a decolorizing agent causes dehydration of the multi-layered peptidoglycan of the cell wall. This causes a decrease in the space between the molecules causing the cell wall to trap the crystal violet iodine complex within the cell. Hence the Gram-positive bacteria do not get decolorized and retain primary dye appearing violet. Also, Gram-positive bacteria have more acidic protoplasm and hence bind to the basic dye more firmly. In the case of Gram-negative bacteria, the alcohol, being a lipid solvent, dissolves the outer lipopolysaccharide membrane of the cell wall and also damages the cytoplasmic membrane to which the peptidoglycan is attached. As a result, the dye-iodine complex is not retained within the cell and permeates out of it during the process of decolonization. Hence when a counterstain is added, they take up the color of the stain and appear pink.

Requirements for Gram stain

1. Compound light microscope
2.  Reagents and glasswares
   Bunsen flame
   Wire loop
   Clean grease-free slides
   Marker pen
   Crystal violet (Basic dye)
   Gram’s iodine(mordant)
   95% ethanol (decolorizing agent)
   1% safranine or dilute carbol fuchsin or neutral
   red
3. Specimen

Preparation of bacterial smear: from liquid culture

1. Take a clean, and grease-free slide for making a smear.
2. Take one or two loopful of the bacterial cell suspension and place them on the slide with a bacteriological loop.
3. Then with a circular movement of the loop, spread the cell suspension into a thin area.
4. Allow the smear to air dry.
5. Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.

Preparation of bacterial smear: from the solid medium

• Take a clean, and grease-free slide for making a smear.
• Take a loopful of 0.85% saline i. e. physiological saline and place it on the Center of the slide.
• With a straight wire touch the surface of a well-isolated colony from the solid media and emulsify in the saline drop forming a thin film.
• Allow the smear to air dry.
• Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.

Procedure

1. Cover the smear with crystal violet and allow it to stand for
   one minute.
2. Rinse the smear gently under tap water.
3. Cover the smear with Gram’s iodine and allow it to stand for
   one minute.
4.  Rinse smear again gently under tap water.
5. Decolorize the smear with 95% alcohol.
6. Rinse the smear again gently under tap water.
7. Cover the smear again gently with safranine for one minute.
8. Rinse the smear again gently under tap water and air dry it.
9. Observe the smear first under the low power (10 X) objective, and then under the oil immersion (100X) objective.
   

Escherichia coli Gram-stained smear under a microscope
Rod-shaped
pink in color
that’s why Gram-negative Bacilli

Bacillus species growth on CLED agar and gram stain as shown below-

Gram-positive cocci in pairs, short chains, and long chains under a microscope

Gram-positive cocci under microscope | Gram-positive bacteria | GPC in pair, chain, and cluster possible organism (Staphylococcus aureus) as shown below-

Yeast cell identification-
Colonial morphology
on blood agar from urine specimen

Specimen: High Vaginal Swab (HVS)
Heavy growth of Streptobacillus was seen on blood agar from which Gram stain performed and finally result shown in the video.

Diphtheroids | growth on blood agar | gram stain

Urethral discharge /Gram’s negative diplococci/ Neisseria gonorrhoeae /Gonococcus in Gram stain as shown below-

Growth of Candida albicans on  SDA, Gram stain and its germ tube test (GTT) positive as shown below-

A patient was 53 years old with Chronic Otitis Media (COM) having a failure of antibacterial drugs- Pus swab from ear discharge sent to microbiology section for Gram staining- result found – Fungal spores with plenty of pus cells and lacking bacteria as shown in video-

Haemophilus Influenzae in Gram stain is Gram-negative coccobacillus or short bacilli to a long thread-like and pleomorphic forms as shown below-

Pus under the microscope showing Gram-positive cocci in singles, pairs, and clusters।। Staphylococcus as shown below-

Ziehl- Neelsen /AFB Staining

Principle

Presence of higher alcohol, glycerol, fatty acid, and especially mycolic acid
in the cell wall have been found responsible for keeping the acid-fast
property of bacteria.

Requirements

• Compound light microscope
• Reagents and glasswares
  Bunsen flame
  Wire loop
  Clean grease-free slides
  Marker pen
  Sprit lamp
  Carbol fuchsin
  20% Sulphuric acid
  Methylene blue
• Specimen

Procedure

1.  Make smear on a clean glass slide.
2. Dry and fix the smear.
3. Cover the smear with a strong carbon fuchsin solution.
4. The heat from underneath the slide until just steam comes from the stain. Do not boil.
5. Wait for five minutes.
6. Rinse with water.
7. Decolorize by 20% Sulphuric acid or 3% acid alcohol until the smear becomes pale pink in color. (wait for nearly five
   minutes)
8. Rinse with water.
9. Counterstain with methylene blue for one minute.
10. Rinse with water.
11. Drain and dry.
12. Observe the smear first under low power (10 X) objective, and
    then under oil immersion (100 X) objective.

Acid-fast staining | AFB stain | Z -N staining a fully practical microbiology | AFB positive

Observation of Acid Fast Bacilli(AFB) /Z- N stained slide under the microscope showing pink, beaded, thin slender rod with sometimes curved having size about 1 -8 x 0.2 -0 .6 µm i.e Mycobacterium tuberculosis as shown below-

Acid alcohol Fast stained slide of Mycobacterium tuberculosis on counter stain methylene blue showing pink, beaded, thin slender rod with sometimes curved having size about 1 -8 x 0.2 -0 .6 µm as shown below-

Auramine phenol stain/ Mycobacterium/ positive/ Fluorescence microscope as shown below-

Modified Ziehl-Neelsen stain-

Modified Ziehl-Neelsen for Leprosy causative agent Mycobacterium leprae as shown below-

Mycobacterium leprae on microscope/cold Ziehl-Nelsen stain/afb Positive:
Red solid bacilli, some are beaded forms, occurring single also in masses i.e. globi and thus grade is  6+ because more than 1,000 bacilli , clumps and globi in every field as shown below-

Various stages of Cyclospora cayetanensis in Modified Ziehl-Neelsen stained smear as shown below-

Nocardia asteroides under acid-fast stained slide

Kinyoun’s stained slide 0f Cryptospoidium parvum under the Microscope

Albert’s Staining

It is used to stain Corynebacterium diphtheriae causative agent of diphtheria.

Capsule Staining

It is used to stain capsules of organisms like the fungus Cryptococcus neoeormans and bacteria Streptococcus pneumoniae , Klebsiella pneumoniae, etc.

Spore staining

It is used to stain spores.

Negative staining

Negative staining is so-called because the background gets stained and the organism remains colorless. e.g.  India ink, nigrosin, or eosin are the stains used. Here below. you can see capsules of Cryptococcus neoformans in nigrosin preparation.

Cryptococcus capsules in Nigrosin preparation of CSF as shown below-

Capsulated Klebsiella pneumoniae from drain sample under India ink preparation as shown in video-

CSF I Cryptococcus India ink Positive I KOH I Nigrosin I  LPCB I Gram stain I Giemsa  I chlorazole black E as shown below-

LPCB stain

Introduction of LPCB stain

LPCB stain stands for lactophenol cotton blue and it is a combination of fixative, staining, and clearing agent. LPCB uses both as a mounting fluid and a stain. This is used for staining and microscopic identification of fungi. Its contents functions are as follows-

Lactic acid: It helps in preserving the morphology of the fungal elements.

Phenol: It acts as a disinfectant.

Cotton blue: It stains the fungal elements as well as intestinal parasitic (cyst, ova, and oocyst) and non-parasitic structures (vegetable cells, mucus, muscle fibers, and other artifacts).

Glycerol: It is a hygroscopic agent that prevents drying.

Principle of LPCB stain

Ingredients of LPCB stain like lactic acid acts as a clearing agent and aids in preserving the fungal structures. Similarly, phenol kills the organism and fixes it while glycerol prevents drying. Cotton blue stains the chitin in the cell wall of fungi and identification of filamentous fungi is made by their characteristic microscopic morphology such as shape, size, arrangement of spores, and hyphae providing color to the structure. It can be used alone or in conjunction with KOH.

Composition of LPCB stain 

For 50 ml
Lactic acid : 10 ml
Phenol : 10 ml
Glycerol :20 ml
Cotton blue (Poirier blue or Aniline blue): 0.025 g
Distilled water : 10 ml

• Dissolve phenol in lactic acid, glycerol, and distilled water.
• Finally, add cotton blue and mix well.
• But this LPCB stain is prepared over two days.
• On the first day, dissolve the cotton blue in the distilled water and leave it overnight to eliminate insoluble dye.
• On the second day, wearing gloves add the phenol crystals to the lactic acid in a glass beaker. Place on a magnetic stirrer until the phenol is dissolved or do manually.
• Add the glycerol.
• Filter the cotton blue and distilled water solution into the phenol/glycerol/ lactic acid solution.
• Mix and store at room temperature.

Requirements for test

• Compound light microscope
• LPCB stain
• Clean and grease-free microscopic slides
• Coverslip
• Dropper or bamboo sticks
• Fungal growth  in the medium

The procedure of LPCB preparation

• Take a clean and grease-free glass slide.
• Put a large drop of LPCB with a Pasteur pipette or dropper.
•  Transfer a small quantity of the culture to the drop.
• Tease the culture (in case of a mold) well with teasing needles
  so as to get a uniform spread.
• Put on a coverslip gently to avoid entrapment of air bubbles.
•  Examine under low- (10 X) and high-power (40 X) objectives.
• Observe the morphological features carefully as shown below.
  

  Observation

  Fungi appear as dark blue stained mycelium.

  Results and interpretations

  Different fungi under LPCB wet mount will show different types of morphological structures including hyphae and spores. We concern with Aspergillus as shown below.

• Fungal spores, hyphae, and fruiting structures: Takes stain blue
• Background: stains pale blue.
  

  Application of LPCB stain

•  For staining and microscopic identification of fungi observing fungal spores, hyphae, and fruiting structures.
• It is also applicable in parasitology for the observation of Cyst of intestinal protozoa and ova takes blue color while ova of helminths are stained deep blue.
• Various fungi and their structures ( yeast cells, budding yeast, hyphae, pseudohyphae, mycelium, spores) in LPCB preparation are as follows-

Aspergillus fumigatus Colony on SDA, LPCB tease mount under microscopy

Trichosporon on SDA and lactophenol cotton blue preparation under the microscope

Geotrichum growth on SDA and its fungal structures on lactophenol cotton blue preparation

Bipolaris growth on SDA and its structures on lactophenol cotton blue preparation

Syncephalastrum in lactophenol cotton blue preparation under the Microscope

Penicillium colonial morphology and its microscopic features in lactophenol cotton blue tease mount under microscope

Fungus, Acremonium on SDA and lactophenol cotton blue preparation

Aspergilus flavus on Czapek Dox agar, Cornmeal agar, and lactophenol cotton blue tease mount

Fusarium growth on SDA and its structures in lactophenol cotton blue preparation

Cryptococcus neoformans in lactophenol cotton blue tease mount

Candida albicans in LPCB tease mount

Cladosporium on SDA and its fungal structures on lactophenol cotton blue preparation

LPCB Mount of Curvularia species

Mucor in LPCB mount

Lactophenol cotton blue tease mount procedure and observation under the Microscope

Sporothrix schenckii under the microscope in lactophenol cotton blue preparation showing the following structures-conidia, conidiophores, and septate hyphae , Conidia in clusters

Trichophyton mentagrophyte Isolated: features-
Helical pattern on lactophenol cotton blue Mount seen
Urease test-Positive
Hair perforation test-Positive

Arthroconidia of Trichosporon inkin – Long Cylindrical in Shape

Bioburden determination of different substances used in pharmaceuticals

…

Microbial monitoring of the environment of pharmaceutical industries
…

Water quality testing

…

Air quality evaluation

….

Floor evaluation

…

Validation of Autoclave. Hot air oven and incubator

….

Sterility testing of different pharmaceutical products-Infusion, Injection, and eye drops

…

Antibiotic Susceptibility Testing: Disc Diffusion methods

Antimicrobial  Susceptibility Testing (AST)

Antimicrobial Susceptibility Testing (AST) is a laboratory method for determining the susceptibility of organisms to therapy with antibiotics. Antimicrobial Susceptibility Testing (AST) is usually carried out to determine which antibiotic / antimicrobial agent will be most successful in treating a microbial infection in vivo.

Antimicrobial Susceptibility Testing Methods

• Disc diffusion method e.g. Kirby-Bauer  and Stokes method
• Dilution method

1.  Tube dilution
2. Agar dilution
3. Disc diffusion and Dilution method e.g. Epsilometer Test or  E-Test

Preparation of the inoculum

The routinely used method is the turbidity standard (0.5 McFarland).

Emulsify 2-3 colonies in sterile saline matching the turbidity standard.

Kirby-Bauer Method (Disc Diffusion Test)

Commonly used method for determining the antibiotic susceptibility of a bacteria.

Test Requirements

• Pure cultures of the organism isolated from the clinical specimen
• Muller Hinton Agar
• Antibiotic Disks
• Turbidity Standard
• Swabs

Antimicrobial Susceptibility Testing Procedure

1. Mostly Muller Hinton agar( MHA) is used in this antibiotic susceptibility test.
2. Emulsify 2-3 colonies in sterile saline matching the turbidity that standard (0.5 McFarland).
3. Place a sterile cotton swab in the bacterial suspension and remove the excess fluid by pressing and rotating the cotton against the inside of the tube above the fluid level.
4. The swab is streaked in three directions over the surface of the MHA  to obtain uniform growth.
5. Allow the plates to dry for 10-15 minutes.
6. Using sterile forceps or a suitable disk dispenser, place paper disks impregnated with a fixed concentration of an antibiotic, on the surface agar plates having distance disc to disc 25 mm while plate border to disk 15 mm.
7. Incubate the plates at 37°C for 24 hours.
8. Following overnight incubation, measure the diameter of the zone of inhibition in millimeters (mm) around each disc.

Result Interpretation of Susceptibility Testing 

Using a standard table of antibiotic susceptibilities, determine if the strain is resistant, intermediate, or susceptible to the antibiotics tested.

Determination of minimum inhibitory concentration (MIC and MBC) of antibiotics and other antimicrobial compounds by tube and agar diffusion methods: E- test

Dilution method

Used to determine the minimal concentration of the antimicrobial agent to inhibit or kill microorganisms.

Methods: It can be achieved by following methods-

1. Tube dilution methods
2. Agar dilution method

Minimum inhibitory concentration (MIC): The lowest concentration of antimicrobial agent that inhibits the growth of organisms.

Minimum bactericidal concentration (MBC): Lowest concentration of an antimicrobial that kills organisms isolated from the patient.

Tube dilution methods

1. In this method, sterile Muller Hinton broth (MHB) is used.
2. Two folds dilution of antibiotics in the broth i.e. 2µg/ml, 4µg/ml, 8µg/ml, 16µg/ml, and so on is made.
3. Then broth culture (0. 1 ml ) of test organism is added to the prepared dilutions.

Method

There are two types of dilution methods by which it can be achieved and they are-

a) Micro-dilution method

It is performed in 96 well microtiter plates.

About 0.1 ml total broth volume is used.

b) Macro-dilution method

• Test tubes for this test are used.
•  About 1 ml total broth volume is used in which two-fold dilution of antibiotics made.

Incubation

In both dilution methods, after adding the test organism, the tubes are incubated at 37°C for 24 hours.

Result Interpretation

The minimum inhibitory concentration (MIC) of antibiotics is determined in µg/ml.

 Minimum inhibitory concentration (MIC): Minimum concentration of antibiotic that inhibits the growth of bacteria i.e. a clear broth.

Minimal bactericidal concentration (MBC): The minimal bactericidal concentration can be determined by sub-culturing all tubes showing no visible turbidity. The tube with the highest dilution that fails to yield growth on the subculture plate contains the MBC of antibiotics for the test strain.

Agar dilution method

Serial dilution of antibiotics is prepared in agar and poured into plates.

Epsilometer test or E-test

1. It is a quantitative assay for determining the Minimum Inhibitory Concentration (MIC) of antimicrobial agents against microorganisms and for detecting the resistance mechanisms.
2. MIC Test Strip are paper strips with special features that are impregnated with a predefined concentration gradient of antibiotics.
3. On one side of the strip is indicated a MIC scale in μg/ml and a code that identifies the antimicrobial agent.
4. The exponential gradient of the antimicrobial agent is immediately transferred to the agar matrix.
5. After 18 hours of incubation or longer, an asymmetrical inhibition ellipse centered along the strip is formed.
6. The MIC is read directly from the scale in terms of μg/ml at the point where the edge of the inhibition ellipse intersects the MIC Test Strip as shown above picture.

Epsilometer test or E-test Procedure

1. Take 24-48 hours old broth (Liquid) culture of bacteria to be tested.
2. Place a sterile cotton swab in the bacterial suspension and remove the excess fluid by pressing and rotating the cotton against the inside of the tube above the fluid level.
3. The swab is streaked in three directions over the surface of the Mueller-Hinton agar to obtain uniform growth. A final sweep is made around the rim of the agar.
4. Allow the plates to dry for five minutes.
5. With the help of sterile forceps apply E-test strips at equal distance on inoculated Muller Hilton agar plate.
6. Incubate the plates at 37°C for 24 hours.
7. Following overnight incubation, an inhibition ellipse is produced.
8. The edge of the ellipse corresponding to the antibiotic concentration on the scale indicates the MIC.

Result Interpretation

MIC:  the point where the edge of the inhibition ellipse intersects the MIC Test Strip i.e. 0.75 μg/ml.

Advantages

• Simple
• Active
• Reliable

Factors Influencing Antimicrobial Susceptibility Testing

pH: The pH of each batch of MHA should be checked when the medium is prepared.  The exact method used will depend largely on the type of equipment available in the laboratory. The agar medium should have a pH between 7.2 and 7.4 at room temperature after gelling. If the pH is too low, certain drugs will appear to lose potency (e.g. aminoglycosides, quinolones, and macrolides), while other agents may appear to have excessive activity (e.g. tetracyclines).  If the pH is too high, the opposite effects can be expected.

Moisture: If, just before use, excess surface moisture is present, the plates should be placed in an incubator (35°C) or a laminar flow hood at room temperature with lids ajar until excess surface moisture is lost by evaporation (usually 10 to 30 minutes).  The surface should be moist, but no droplets of moisture should be apparent on the surface of the medium or on the Petri dish covers when the plates are inoculated.

Effects of Thymidine or Thymine: Media containing excessive amounts of thymidine or thymine can reverse the inhibitory effect of sulfonamides and trimethoprim, thus yielding smaller and less distinct zones, or even no zone at all, which may result in false-resistance reports.  MHA that is as low in thymidine content as possible should be used.  To evaluate a new lot of MHA, E. faecalis ATCC 29212, or alternatively, E. faecalis ATCC 33186, should be tested with trimethoprim/sulfamethoxazole( Co-trimoxazole) disks.  Satisfactory media will provide essentially clear, distinct zones of inhibition 20 mm or greater in diameter.  Unsatisfactory media will produce no zone of inhibition, growth within the zone, or a zone of less than 20 mm.

Effects of Variation in Divalent Cations: Variation in divalent cations, principally magnesium and calcium, will affect the results of aminoglycoside and tetracycline tests with Pseudomonas aeruginosa strains.  Excessive cation content will reduce zone sizes, whereas low cation content may result in unacceptably large zones of inhibition. Excess zinc ions may reduce zone sizes of carbapenems.  Performance tests with each lot of MHA must conform to the control limits.

Testing strains that fail to grow satisfactorily: Only aerobic or facultative bacteria that grow well on unsupplemented MHA should be tested on that medium.  Certain fastidious bacteria such as Haemophilus species, Neisseria gonorrhoeae, Streptococcus pneumoniae, and viridans and ß-hemolytic streptococci do not grow sufficiently on unsupplemented MHA. These organisms require supplements or different media to grow, and they should be tested on the media described in separate sections.

Key Notes on Antimicrobial Susceptibility Testing (AST)

1. Ideal antibiotic therapy is based on the determination of the etiological agent and its relevant antibiotic sensitivity.
2. Empiric treatment is often started before laboratory microbiological reports are available when treatment should not be delayed due to the seriousness of the disease.
3. Genotypic AST Methods: PCR, DNA microarray and DNA chips, and loop-mediated isothermal amplification (LAMP) are some of the genotypic techniques for the detection of antibiotic resistance.
4. Emerging Methods for AST:  Microfluidics-based diagnostics are one of the most promising emerging tools for AST. Microfluidics is an evolving field characterized by the manipulation of fluids in micro-volume, thereby offering portability, cost-effectiveness, multiplexing, reproducibility, and a controllable environment in an in vitro system.
5. The newly developed MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) is a more straightforward and cost-effective modulation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) used for both AST and MIC determination.
6. Clinical and Laboratory Standards Institute (CLSI) and  European Committee on Antimicrobial Susceptibility Testing (EUCAST) are Institutions those AST guidelines followed globally.

Related Video on Antimicrobial Susceptibility Testing 

#Antimicrobial Susceptibility Testing ||Kirby Bauer Disc Diffusion Method|| AST-

#E-strip and antibiotic disks putting easiest and fastest method।।AST।। Antimicrobial Susceptibility Testing –

#Bacterial inoculation swabbing and application of antibiotics disks-

#How to test MIC of antibiotics for bacteria-

Testing efficacy of disinfectants and preservatives in Pharmaceutical  Microbiology 

Introduction of disinfectant

Disinfection is the process of elimination of most pathogenic organisms excluding bacterial spores on inanimate objects. The efficiency of a disinfectant is tested by measuring the rate of killing effect against a range of microorganisms under specified conditions.

Characteristics of  ideal disinfectant 

1. A wide spectrum of activity
2. Active at any pH
3. Stable
4. High penetrating power
5. Long shelf life
6. Able to destroy microorganisms
7. No bad odor within a particular time
8. Speedy in action
9. Efficacy shouldn’t be lost on reasonable dilution.
10. Non-toxic or  non-allergic or non-corrosive or non-irritant
11. Be active in the presence of organic matter

Factors influencing the effectiveness of a disinfectant

• Nature of the item to be disinfected
• Number and resilience of the contaminants
• Amount of organic material present
• Type and concentration of disinfectant
• Duration and temperature of exposure

Why testing of disinfectants is necessary?

• To know the required effective dilution.
• To know the time taken for the onset of action.
• Periodical monitoring of its activity.
• As disinfectants are known to lose their action on long-standing and in the presence of organic matter their efficacy must be tested periodically

Various types of testing disinfectant

Testing for the potency of disinfectants including-

1. Koch method
2. Minimum inhibitory concentration (MIC) test,
3.  Rideal-Walker test,
4. Chick-Martin and Garrod test,
5. Capacity use dilution test,
6. Stability test,
7. Test for disinfecting action on surfaces and
8. In-use test

Koch method

Spores of Bacillus anthracis are dried on silk thread and are subjected to the action of disinfectants. Later, it is washed and transferred to a solid medium.

Minimum inhibitory concentration (MIC) test

This test measures the lowest concentration of the disinfectant that will inhibit the growth of a known strain of bacterium.

Rideal-Walker test

The test compares the bactericidal activity of the disinfectants with that of phenol. It measures the phenol coefficient of the disinfectants under the standard condition.

Chick-Martin and Garrod test

This assay stimulates natural conditions more closely than the Rideal-Walker test. In this test, a standard amount of organic matter is incorporated into the test mixture. The use of feces has several objectives but Garrod has devised a modification of the Chick-Martin test with the use of yeast instead of feces.

Capacity tests

Place each time soiled instrument into a container with disinfectant and add a certain quantity of dirt and bacteria to the solution. The ability to retain activity in the presence of an increasing load is the capacity of the disinfectant. In a capacity test,  the disinfectant challenges repeatedly by successive additions of bacterial suspension until its capacity to kill has been exhausted. These tests simulate the practical situations of housekeeping and instrument disinfection. The most common and best capacity test is the Kelsey-Sykes test.

Stability test

Stability test measures the stability and long-term effectiveness of the diluted disinfectant in the clean and dirty medium. It uses to supplement the information obtained from capacity use dilution tests.

Test for disinfecting action on surfaces

This test uses to evaluate new disinfectants, does not take into account the effects of detergents or of the mechanical cleaning process.

In use test

In use, the test determines the number of living bacteria, if any, in a sample of disinfectant from any place or container in which it is being used.

Test requirements for In-use test

• Testing disinfectant
• Nutrient agar
• Incubator
• Pipettes and tips

Test procedure

1. Transfer 1 ml of the used disinfectant into 9 ml nutrient broth in a sterile container using a sterile pipette.
2. Place 20 µl drops of this mixture onto 10 different areas of each of two nutrient agar plates.
3. Incubate on a plate for 3 days and other for 7 days at room temperature.
4. Examine the plates and score growth from each drop.

Result Interpretation

Working disinfectant: If growth occurs less than 5 drops out of 10 drops.

Failure of disinfectant: If growth occurs in equal and more than  5 drops out of 10 drops.

Antigen-Antibody Reaction Based Tests 

Introduction of Antigen-Antibody Reaction

Antigen-antibody reaction ( interaction between antigen and antibody) is reversible, occurs at the surface and there is no denaturation of antigen (Ag) and antibody(Ab) during the reaction. Richard J. Goldberg gave the first correct description of the antigen-antibody reaction at the University of Wisconsin in 1952 and it came to be known as “Goldberg’s theory” (of Ag-Ab reaction).  This reaction is highly specific, and an antigen reacts only with antibodies produced by itself or with closely related antigens. The antibody recognizes molecular shapes (epitope, an antigenic determinant) on the antigen. The affinity of the antibody for the antigen is one of the most important features in determining antibody efficacy in vivo. The attachment between antigen and antibody includes various non-covalent interactions between epitope and variable region (VH/VL) domain of an antibody. Chemical bonds responsible for the antigen-antibody reaction are electrostatic bonds, hydrogen bonding. hydrophobic interactions and Van der Waals bonds. Factors affecting Ag-Ab reactions are temperature, pH, and Ionic strength. The strength of antigen-antibody interactions is determined by the following terms-

Affinity: It is the combined strength of total on-covalent interactions between a single antigen-binding site of antibody and a single epitope is the affinity of the antibody for that epitope.

Low-affinity antibody: It binds antigen weakly and dissociates readily whereas high-affinity Ab binds antigen tightly and remains bound longer.

Avidity: It is the strength of multiple interactions between multivalent antibody and antigen and it is better to measure the binding capacity of antibody than affinity. High avidity can compensate for low affinity.

Cross-reactivity: Antibodies elicited by one antigen can cross-react with unrelated antigens if they share identical epitopes or have similar chemical properties.

Types of Antigen-Antibody Reaction

1. Precipitation
2. Flocculation
3. Agglutination
4. Complement Fixation Test
5. Neutralization Tests
6. Radio – Immunoassay  (RIA)
7. Enzyme Immuno Assay (EIA)
8. Immunochromatography
9. Immunoelectrophoresis
10. Immuno Blotting technique
11. Immunofluorescence

Precipitation reaction

When soluble antigen combines with its antibody in the presence of electrolyte forms insoluble (visible) precipitates.  This type of antigen-antibody reaction is more sensitive for antigen detection. e.g. Elek’s test (diphtheria toxin), Biken test (to detect enterotoxigenic E. coli)

The antigen can be detectable both qualitatively as well as quantitatively.

It is more useful for antigen detection to as least as 1µg.

Flocculation

It is a special type of precipitation where precipitates remain suspended instead of sedimentation. e.g. VDRL, Kahn test (a tube flocculation test for syphilis), RPR

Agglutination

Antigen-antibody reaction in which antibody combines with a particulate antigen such reaction called agglutination. It is more sensitive than precipitation for antibody detection. It is further subcategorized into following types-

• Slide agglutination
• Tube agglutination
• Indirect passive haemagglutination
• Reverse passive haem-agglutination
• Antiglobulin ( Coomb’s) test

• Slide agglutination:-serotyping e.g. blood grouping ( ABO and Rh typing)  and cross match ( major and minor)
• Tube agglutination: Widal test, Weill- Felix reaction, Paul Bunnel test.
• Indirect passive haemagglutination:

1. Where antibodies are coated on RBC
2. Turkey red cells are often used as carrier particles because they are nucleated and sediment rapidly.
3. Red cells coated with antigens are called sensitized cells e.g. TPHA

• Reverse passive haem-agglutination:- The technique utilizes stabilized red cells coated with specific viral antibodies.
• Antiglobulin ( Coomb’s) test: It is applicable for the detection anti-Rh Ab and incomplete antibody of brucellosis. It is of two types, direct Coomb test (DCT) and indirect Coomb test (ICT). DCT is applicable to assist diagnose the cause of RBC destruction (hemolytic anemia); investigating a blood transfusion reaction; to diagnose hemolytic disease of the newborn (HDN). ICT uses to determine whether there are antibodies to the Rh factor in the mother’s blood in a case of hemolytic disease of new-born (HDN).

Complement fixation test

Complement fixation test brief form is CFT. Complement is a nonspecific protein found in our normal serum. This CFT was extensively used for the diagnosis of syphilis caused by Treponema pallidum, a serological test called Wassermann test or Wassermann reaction named after the introducer surname bacteriologist August Paul von Wassermann in 1909. The whole complement system is made up of nine components i.e. complement one ( C1)to C9 Complement proteins are heat-labile and are destroyed by heating at 56°C for 30 minutes. Complement binds to antigen and antibody  ( Ag-Ab) complex. When the antigen is an RBC it causes lysis of red blood cells ( RBCs). It has the ability to screen against a large number of viral and bacterial infections at the same time.

Neutralization Tests

When antitoxin combines with its corresponding toxin, neutralization occurs. Few examples of these tests are the Schick test (diphtheria)  and the Dick test ( Streptococcus pyogenes).

 Radio – Immunoassay  (RIA)

• Major analytes  up to picogram (10 –¹² g) quantities
• The most common label used is radio Isotopes and measured by gamma-spectrometer.
• Used for quantification of hormones, drugs, tumor marker, viral antigen, etc.

Enzyme Immuno Assay (EIA)

It measures enzymes labeled antigen, antibody and it may be homogenous or heterogeneous. Major types of heterogenous EIA is enzymes linked ImmunoSorbent Assay (ELISA)

 ELISA ( enzymes linked ImmunoSorbent Assay)

This technique involves the use of an enzymes system and an immunosorbent ( an absorbing material specific for one component of reaction, antibody, or antigen). The absorbing material could be either agarose or a solid matrix ( microwell, membrane).

Enzyme used-

• Horse radish peroxidase
• alkaline phosphates

Substrate used-

• O- Phenyl diamine dihydrochloride (OPDDH)- for peroxidase
• P- nitrophenyl phosphate (PNP) -for alkaline phosphatase

Modification of ELISA

• Indirect ELISA:-  detect antibody  → Antigen is coated on the plate surface.
• Direct ELISA / Sandwich ELISA: detect antigen→ Antibody is coated on the plate surface.

Immunochromatography

Immunochromatography is an association between chromatography and immunoassay ( antigen-antibody reaction). This technology is widely applicable in laboratory medicines for the rapid detection of antigens (various microbes) and antibodies. Depending on the test, either antigen (for detection of antibody) or antibody (for detection of antigen) are immobilized on a nitrocellulose membrane in discreet spots in a cartridge device. Following antibody or antigen capture and a flow-through wash step, sequential addition of an enzyme-labeled conjugate and substrate results in the appearance of a colored reaction product in the form of bands or spots directly on the membrane. e.g.  rapid/ quick/ spot tests of HIV, HCV antibody tests, HBsAg test

Immuno Blotting technique

• Southern blotting – detect DNA
• Northern blotting  – detect RNA
• Western blotting – defection protein ( confirmatory test of HIV)
• Eastern blotting- to analyze protein post-translational modifications (PTMs)

Immunoelectrophoresis

Immunoelectrophoresis is a combination of immuno-diffusion and electrophoresis. It is a special type of precipitation that occurs in agar under an electric field. Initially, an antigen mixture is separated by electrophoresis and then tested by double immuno-diffusion.

Immunofluorescence

Immunofluorescence also called cell imaging technique is a combination of Immuno and fluorescence and this assay is used primarily on biological samples and is classically defined as a procedure to detect antigens in cells using antibodies. The specificity of the antibody to its antigen is the base for immunofluorescence.

Application of Antigen-Antibody Reaction

These Ag-Ab reaction-based tests are widely used in laboratory techniques for a serological test of blood compatibility and various pathogenic infections.

1. Tests based on precipitation principle are Elek’s test (diphtheria toxin), Biken test (to detect enterotoxigenic E. coli)
2. Flocculation based tests are VDRL, Kahn test (a tube flocculation test for syphilis), RPR
3. Assay belonging to agglutination are blood grouping, X-match, Widal test, Weill- Felix reaction, Paul Bunnel test, TPHA, Coomb’s tests ( direct and indirect)
4. Complement fixation test (CFT) uses to screen a large number of viral and bacterial infections at the same time.
5. Neutralization tests are Schick (diphtheria)  and Dick test ( Streptococcus pyogenes).
6. RIA uses for the quantification of hormones, drugs, tumor markers, and viral antigens.
7. ELISA is widely applicable to detect antigens and antibodies of various microorganisms.
8. Immunochromatographic test- This test is widely adopted as the quick or spot or screening test for laboratory diagnosis of various diseases etiological agents as well as the conditions and they are-

• Detection of toxins
• Pregnancy tests- detection of human chorionic gonadotropin (hCG)
• Diagnosis of parasitic infections-Malaria rapid test, ImmunoCardSTAT for Cryptosporidium/Giardia
• Diagnosis of bacterial infections-ImmunoCard Mycoplasma, which detects Mycoplasma pneumoniae specific IgM in serum samples, ImmunoCardSTAT SpSA for H. pylori antigens in stool, Vibrio cholerae O1 and O139 from stool specimens, Binax NOW Streptococcus pneumoniae antigen card – Streptococcus pneumoniae antigen detection in CSF or in urine
• Diagnosis of viral Infections-Antigen detection e.g. Binax NOW RSV, Remel Xpect RSV, ImmunoCardSTAT! For Rotavirus, Influenza A/B (BinaxNOW Influenza A & B), Dengue NS1, SARS-CoV-2 antigen, HBsAg test. Antibodies detection e.g. detection of HIV-1 and HIV-2 antibodies (Tridot), Dengue IgM and IgG, SARS-CoV-2 IgM, and IgG

9.Immunoelectrophoresis is used to detect normal as well as abnormal proteins, such as myeloma proteins in human serum, M-proteins in serum and urine.

10. Immuno Blotting technique uses to detect DNA, RNA, protein, and PTMs.

11. Immunofluorescence is a type of immunohistochemistry method which utilizes fluorophores to visualize various cellular antigens.

Contd…

Further Reading

1. https://www.britannica.com/science/Bunsen-burner
2. https://www.sciencedirect.com/topics/engineering/bunsen-burner
3. https://www.goodscience.com.au/year-7-science/the-bunsen
4. https://en.wikipedia.org/wiki/Bunsen_burner
5. https://thermolabscientific.com/other-products/bod-incubator/
6. https://thermolabscientific.com/other-products/bacteriological-incubato/
7. http://bcas.du.ac.in/wp-content/uploads/2020/04/Incubators-are-used-for-the-growth-of-microorganisms-in-many.pdf
8. https://pages.binder-world.com/en/co2-incubator-function
9. https://www.fishersci.com/us/en/browse/90088090/co2-incubators
10. https://www.researchgate.net/publication/331899509_Hot_Air_Oven_for_Sterilization_Definition_Working_Principle
11. http://www.acmasindia.com/blog/hot-air-oven/
12. Textbook of Medical Laboratory Technology by Praful B. Godkar, Darshan P. Godkar
13. https://ultrasoniccleanersmfgr.com/hot-air-oven.html gclid=CjwKCAjwps
14. https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3340325
15. https://www.stericox.com/laboratory-oven/hot-air-oven.html
16. https://www.prestogroup.com/blog/use-of-hot-air-oven-in-laboratories/
17. https://en.wikipedia.org/wiki/Hot_air_oven
18. https://www.sciencedirect.com/topics/engineering/autoclave
19. https://en.wikipedia.org/wiki/Autoclave
20. https://tuttnauer.com/blog/autoclave
21. https://www.steris.com/healthcare/knowledge-center/sterile-processing/everything-about-autoclaves
22. https://ehs.princeton.edu/book/export/html/380
23. https://www.cdc.gov/infectioncontrol/guidelines/disinfection/sterilization/steam.html
24. https://www.explainthatstuff.com/autoclaves.html
25. https://www.sciencedirect.com/topics/physics-and-astronomy/electron-microscope/
26. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/light-microscopes
27. https://www.jove.com/v/5041/introduction-to-light-microscopy
28. https://open.oregonstate.education/generalmicrobiology/chapter/microscopes/
29. https://www2.mrc-lmb.cam.ac.uk/microscopes4schools/microscopes2.php
30. https://apps.who.int/iris/bitstream/handle/10665/274382/MM-SOP-12-eng.pdf?sequence=17&isAllowed=y
31. https://micro.magnet.fsu.edu/primer/anatomy/cleaning.html
32. https://labmal.com/product/nutrient-agar-500g/
33. http://himedialabs.com/TD/M001.pdf
34. https://en.wikipedia.org/wiki/Nutrient_agar
35. https://catalog.hardydiagnostics.com/cp
36. https://www.thomassci.com/Laboratory-Supplies/Microbiological-Media/_/Sabouraud-Dextrose-Agar
37. http://himedialabs.com/TD/M063.pdf
38. https://en.wikipedia.org/wiki/Sabouraud_agarhttps://drfungus.org/knowledge-base/sda/
39. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1706009/
40. https://mycology.adelaide.edu.au/laboratory/lacto/
41. http://himedialabs.com/TD/S016.pdf
42. The testing of disinfectants: Gerald Reybrouck, International Biodeterioration and Biodegradation 41 (1998) 269-272
43. Joan F.Gardner, Margaret M Peel. 1991. Introduction to sterilization and disinfection control, 2ndedition, Churchill Livingstone
44. https://www.microrao.com/micronotes/pg/testing_of_disinfectants.pdf
45. https://www.slideshare.net/naik4naik/disinfection-testing-for-disinfection
46. https://pubmed.ncbi.nlm.nih.gov/2567307/
47. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581910/
48. https://www.sigmaaldrich.com/technical-documents/articles/biology/generation-of-antibodies.html
49. https://link.springer.com/chapter/10.1007%2F978-3-642-70393-5_7
50. https://www.frontiersin.org/articles/10.3389/fimmu.2013.00302/full
51. https://www.brainkart.com/article/Precipitation-Reactions–Test,-Application_20189/
52. https://immunochemistry.com/2016/07/07/back-basics-immunoassay/
53. https://labtestsonline.org/tests/direct-antiglobulin-test