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Microbiology Practical: Bachelor of Nursing and B.Sc. Nursing

The topic ‘Microbiology Practical’ is a complete solution package for both Bachelor of Science (B.Sc.) in Nursing and Bachelor of Nursing (B.N.). All the contents of this microbiology practical are with the description as well as a visual demonstration.

  Contents of Microbiology Practical

1. Study of the compound microscope
2. Identify the different culture media, its composition, and classification
3.  Application of different sterilization methods
4.  Preparation of smear from solid/liquid media
5. Gram staining of the prepared and fixed smear of a given culture.
6. Study of colony characteristics of bacteria on different media plates.
7. Demonstration of colony characteristics of lactose fermenting (LF) and non-lactose fermentation (NLF) Colonies on MacConkey agar plates, hemolytic/non-hemolytic colonies on blood agar plates, and colonies on nutrient agar.
8. Ziehl-Neelsen (Z-N) staining for acid-fast bacilli
9. Observation of ZN stained standard slide.
10. Study of fungi ( yeast and mold)
11. Observation of standard slide of Gram-stained Candida
12.  Bacteriological examination of urine samples, antibiotic sensitivity test of isolated organisms from urine samples.
13. Saline/iodine preparation of stool samples for microbiological examination.
14.  Microbiological examination / biochemical tests of vaginal swabs
15. Demonstration of VDRL/RPR test, WIDAL test
16.  Microbiological examination, biochemical tests of pus sample from the wound.
17. Microscopical examination of ova of common intestinal parasites.
18. Study of blood slides for parasite examination.

Microbiology Practical Lists for Nursing Students

#1. 0 Study of compound microscope#

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 center 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 purpose of the microscope is 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 the 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.

#Study of compound microscope ||Practical based explanation|| Microbiology#

Sizes of Microorganism

Bacteria

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

Viruses

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

Parasites

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

Helminthes sizes are variable-

Cestodes 1mm to several meters in length

Nematodes vary in size from  5mm to even 1 meterFungus

Yeast like appearance 2-30µm

Mould appearance 2-5 µm

Spherical like appearance 5-300µm

Handle and  care  of the microscope

Handle with care

Most microscope problems occur as a result of improper handling. When carrying your microscope, 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 a microscope bag.

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

Keep lenses clear of slides

When using your microscope 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 your microscope covered when not in use even if the microscope 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 the microscope 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

Microscope 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.

#2. 0 Identify the different culture media, its composition, and classification#

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 jellifying capacity as 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 maybe 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.

# Culture media the simplest way of identification | Blood |MacConkey | Chocolate |RCM| Nutrient agar-

# 3.0 Application of different sterilization methods#

The most common method of sterilization in a microbiology laboratory is a physical method by heat-

• Dry heat- Hot air oven, Bunsen burner
• Moist heat-Autoclave

#3.1:  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 its 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 that 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 do 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: 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 is 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 non-toxigenic 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 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.

#3.2 : 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 is effective 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 the drawing of fresh steam are repeated until the article is heated up to the sterilizing temperature.

Types of autoclaves

According to structure

According to the structures, autoclaves are of the 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 d 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 milliliter. 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 spore 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.

#3.3:  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 its flame in ‘safety mode’ until you need to heat something.

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).

#4.0: Preparation of Smear from Solid/Liquid Media#

#5.0:  Gram staining of a prepared and fixed smear of given culture#

For both topics : 

Introduction of Gram Stain

Gram stain is a differential stain and therefore it uses to differentiate Gram-positive and Gram-negative bacteria. It was devised originally by a Danish bacteriologist, Hans Christian Joachim Gram (1884) as a method of staining bacteria in his laboratory.

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. 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 attaches. As a result, the dye-iodine complex does not retain within the cell and permeates out of it during the process of decolonization. Hence, when a counterstain uses, they take up the color of the stain and appear pink.

Requirements for Gram stain

a) Compound light microscope

b) 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% safranin or dilute carbol fuchsin or neutral red

c) Quality control strains

Positive Control (PC) : Staphylococcus aureus (ATCC 25923)

Negative Control (NC): Escherichia coli (ATCC 25922)

d) Specimen

Preparation of bacterial smear: from liquid culture

• Take a clean, and grease-free slide for making a smear.
• Take one or two loopful of the bacterial cell suspension and place them on the slide with a bacteriological loop.
• Then with a circular movement of the loop, spread the cell suspension into a thin area.
• 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.

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 of Gram Stain

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 safranin 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 (10X) objective, and then under the oil immersion (100X) objective.

Observation of Gram Stain

Positive Control:   violet color, round in shape in single, pairs and cluster

Test: red color and rod in shape

Negative Control: red in color and rod in shape

Result and Interpretation of Gram Stain

Gram-positive: purple or violet color

Gram-negative: Pink or red in color

Cocci: round in shape

Bacilli: rod in shape

Positive Control(PC): Gram-positive cocci in single, pairs and cluster

Test: Gram-negative bacilli

Negative Control(NC):Gram-negative bacilli as shown above image.

 E. coli under microscope|| Gram stain ||Gram Negative bacilli or Gram-negative rods as shown below-

Variety of bacteria under the microscope showing
Gram-positive bacteria, gram-positive cocci in singles, pairs, clusters i.e. Staphylococcus aureus
Gram-positive rods or bacilli
Bacillus species
Gram-negative bacilli or rods
Salmonella Typhi
Diphtheroids
Sputum gram stained smear
having ideal smear
Gram-positive cocci in pairs inside the pus cell
Gram-positive cocci in chains

#6.0 Study of colony characteristics of bacteria on different media plates#

Introduction of Colony morphology 

Colony morphology of bacteria is the most common diagnostic method in bacteriology for isolation and identification of bacteria on the basis of phenotypic characteristics on solid medium for the color, shape, surfaces, size, elevation, edges, opacity and consistency.

Common colony characteristics of bacteria

Colony morphology of bacteria on the basis of the following features:

1. Size
2. Shape
3. Elevation
4. Margin or edge
5. color
6. Surface appearance
7. Density
8. Consistency
9. Hemolysis
10.  Odor
11. Pigmentation

Size

It is of three types on the basis of the diameter of the colony.

Large: Diameter of colony larger than 1 mm

Medium:  1 mm in diameter.

Small: Less than 1 mm in diameter.

Shape

It is of the following types-

• Circular
• Filamentous
• Irregular
• Punctiform
• Rhizoid
• Spindle

Elevation

Elevation of colony is of following types-

• Flat
• Raised
• Convex
• Umbonate
• Dome-shaped
• Umbilicate

Margin

The margin of colony is further  divided into

• Entire
• Undulate
• Lobate
• Erose
• Filamentous
• Curled

Color

Color of the colony are following types-

• Yellow
• White
• Black
• Cream
• Pink
• Red
• Blue
• Red etc.

Surface appearance

The surface appearance of the colony may be-

• Glistening
• Smooth
• Granular
• Dull
• Rough
• Creamy

Density

Colony density is the ability to see through the colony. On this basis is of following types-

• Opaque:  It can not see through the colony.
• Transparent: It can see through the colony.
• Translucent: It can only see with light shining through.

Consistency

Consistency of colony is best observed by picking up a colony with a loop or needle. It is of the following types-

• Butyrous (buttery)
• Brittle
• Viscid (sticky)
• Friable (Crumbles easily)
• Membranous (pliable)

Hemolysis

It is best observable in 5 % sheep blood agar. It is of three types-

• Alpha (α) hemolysis
• Beta (β) hemolysis
• Gamma (γ) hemolysis

Odor

According to smell, it is of the following types-

• Sweetish
• Aromatic
• Fishy
• Seminal
• Others

Pigmentation

Some bacteria produce pigments and are of the following types-

• Golden yellow (Staphylococcus aureus)
• Lemmon yellow (Micrococcus luteus)
• Prodigiosin( pink/red/ magenata): e.g. Serratia marcescens 
• Pyocyanin (bluish-green) e.g. Pseudomonas aeruginosa
• Pyoverdin/fluorescein (greenish-yellow) e.g. Pseudomonas aeruginosa
• Pyomelanin (brown to black pigment) e.g. Pseudomonas aeruginosa
• Pyorubrin (red): e.g. Pseudomonas aeruginosa

#7.0: Demonstration of colony characteristics of lactose fermenting (LF) and non-lactose fermentation (NLF) Colonies on MacConkey agar plates, hemolytic/ non hemolytic colonies on blood agar plates, and colonies on nutrient agar#

Lactose fermenter and non-lactose fermenter bacteria on MacConkey agar and are gram-negative bacteria as shown below video clip-

Blood agar showing alpha, beta and gamma hemolysis | Use of blood agar

Hemolysis on 5% sheep blood agar (BAP) by the various organisms/bacteria:

• Alpha hemolysis/ greenish discoloration around the colonies (due to partial hemolysis 1-2 mm wide)
• Beta hemolysis/a clear, colorless zone of complete hemolysis(2-4 mm)
• Gamma hemolysis/ no hemolysis e.g. α hemolysis by Streptococcus pneumoniae
• β hemolysis by Staphylococcus aureus or Streptococcus pyogenes or Streptococcus agalactiae
• γ hemolysis by Klebsiella pneumoniae or Escherichia coli etc

Nutrient agar

Colony characteristics of Staphylococcus aureus on nutrient agar
Golden yellow
large i.e. greater than 1 mm in size
Smooth
convex
opaque
and easily emulsifiable

# 8.0: Ziehl-Neelsen (Z-N) staining for acid-fast bacilli # 9.0: Observation of ZN stained standard slide#

Introduction of AFB staining

This acid-fast bacilli in brief AFB staining or Ziehl-Neelsen staining method is a modification of Ehrlich’s (1882) method. Its name is from the surnames of German doctors, bacteriologist Franz Ziehl(1859-1926), and pathologist Friedrich Neelsen (1854-1898).

Principle of AFB Staining

The presence of higher alcohol, glycerol, fatty acid, and especially mycolic acid in the cell wall has been found responsible for keeping the acid-fast property of bacteria. Therefore, AFB staining is useful for Mycobacterium tuberculosis, an etiological agent of tuberculosis.

Requirements

a) Compound light microscope

b) Reagents and glasswares

• Bunsen flame
• Wire loop
• Clean grease-free slides
• Marker pen
• Sprit lamp
• Carbol fuchsin
• 20% Sulphuric acid
• Methylene blue

c) Specimens

In the case of primary tuberculosis

• sputum
• bronchial or laryngeal washing
• Gastric lavage when sputum is swallowed as in children

 In miliary tuberculosis

• bone marrow
• Liver biopsy

Tuberculous meningitis

• Cerebrospinal fluid (CSF)

Renal tuberculosis

• urine

d) Quality control strains

• Positive control (PC): Mycobacterium tuberculosis
• Negative Control: Escherichia coli

Procedure of AFB staining

1. Make smear on a clean glass slide.
2. Dry and fix the smear.
3. Cover the smear with a strong carbol 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 the low power (10X) objective, and then under the oil immersion (100X) objective.

Result and Interpretation

AFB: pink or red bacillus

Background:  Blue ( as counterstain used )

Reporting

There are various ways of a reporting system for AFB stainings such as the Center for Disease Control and Prevention (CDC), the World Health Organization (WHO), and the International Union Against Tuberculosis and Lung Disease (IUATLD). The most common and widely accepted classification is IUATLD and according to it as follows.

• No organism seen: Negative
• 1-9/100 OIF ( oil immersion field): Exact number
• 10-99/100 OIF: +
• 1-10/OIF : ++
• 10/OIF: +++

Related Videos-

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

#Mycobacterium under microscope/ AFB positive: Observation of Acid Fast Bacilli(AFB)/Z- N stained slide-Pink , beaded,thin slender rod with sometime curved having size about 1 -8 x  0 .2 -0 .6 µm i.e Mycobacterium tuberculosis

#AFB stained slide of Mycobacterium tuberculosis on counter stain malachite green-

#Practical No 10:  Study of fungi ( yeast and mold)#

General features fungus

• The fungi are a group of eukaryotic organisms.
• They are found in soil, water, air, and decaying matter are the main sources.
• They have a diversity of morphological appearances, depending on the species.
• They are heterotrophic organisms that lack the definite root, stem, and leaves of higher plants.
• Yeast is unicellular whereas mold is filamentous and multicellular.
• Due to the lack of chlorophyll, they are differentiated from the algae and higher plants.
• They are saprophytic or parasitic because of requiring prepared food.
• Having more complex structures and greater size, they differ from bacteria.

They are mainly found in two forms.

a) yeast and

b) Mold

Yeast

Unicellular form

Size

Width: 1-5 µm

Length: 5-30 µm

Shape

Commonly oval shape but some may be elongated or spherical. Each species has a  characteristic shape, but even in pure culture, there is considerable variation in the size and shape of individual cells, depending on age and environment. Non-motile due to lacking flagella or organ of locomotion.

Mold

Molds are multicellular filamentous fungi consist of mycelium and spores. The mycelium is composed of branching filaments called hyphae, which interface to form a mycelium hyphae are usually 2-10 µm composed of an outer tube-like wall surrounding a cavity, lumen which is filled or lined by protoplasm. The protoplasm is surrounded by plasmalemma. The hyphae may be aseptate i.e. without walls or separate with a central pore in each cross wall.

Mycelium has two forms-

a) Vegetative mycelium

b) Reproductive mycelium also called aerial mycelium.

They reproduce by the formation of different types of sexual and asexual spores that develop from the mycelium. Few examples of molds Aspergillus, Dermatophytes, penicillium , Rhizopus.

Dimorphic fungi

They exist as yeasts in the host tissue and the culture at 37°C and hyphal (mycelium) forms in the soil and in the culture at 22-25°C. Most of them cause systemic infection. e.g. Blastomyces dermatitidis, coccidioides immitis, Histoplasma capsulatum.

Related Videos-

#Study of fungi || Yeast and mold-

#A very simple Saline wet mount technique help you to identify yeast cells of candida from bacteria-

# Experiment No 11.0: Observation of standard slide of Gram-stained Candida#

Requirements for Gram stain of Candida

a) Compound light microscope

b) 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% safranin or dilute carbol fuchsin or neutral red

c) Quality control strains

Positive Control (PC) : Staphylococcus aureus (ATCC 25923)

Negative Control (NC): Escherichia coli (ATCC 25922)

d) Specimen ( Candida growth on SDA)

Preparation of smear

• 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 of Gram Stain

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 safranin 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 (10X) objective, and then under the oil immersion (100X) objective.

Observation of Gram Stain

Positive Control:   violet color, round in shape in single, pairs and cluster

Test: red color and rod in shape

Negative Control: red in color and rod in shape

Result and Interpretation of Gram Stain

Gram-positive: purple or violet color

Gram-negative: Pink or red in color

Cocci: round in shape

Bacilli: rod in shape

Positive Control(PC): Gram-positive cocci in single, pairs and cluster

Test: Gram-negative bacilli

Negative Control(NC): Gram-negative bacilli as shown above image.

Related Videos-

#Observation of standard slide of Gram-stained Candida species|| Yeast under the microscope-

Candida growth on SDA

Smear prepared, air-dried, and heat-fixed.

Finally, Gram-stained performed and focused at 10x objective and finally observed at 100X objective i.e. Oil immersion fields 1000 times greater than its actual size.

Yeast cells of Candida unicellular generally oval in shape but some may be elongated or spherical. hypervariable size of width 2-5 micrometer whereas length 5-30 micrometer.

#Candida albicans colony characteristics on  SDA, Gram-stained smear, and germ tube test (GTT)- Positive as shown below video-

#Experiment No 12: Bacteriological examination of urine samples, antibiotic sensitivity test of isolated organisms from urine samples#

 Introduction of urine culture and sensitivity

A urine culture test is useful to grow and identify organisms, mainly bacteria, and fungi that may cause a Urinary Tract Infection (UTI). Bladder urine is normally sterile and free from any organisms. UTIs are common in females and children than in adult males. The antibiotic sensitivity test  (AST)  helps to select an appropriate antibiotic that is effective against specific types of bacteria or fungi causing any infection.

When need Urine Culture and sensitivity Test?

Following conditions that are useful for culture and sensitivity are-

• when you’ve experienced pain during urinating, less urine, and also frequent urine symptoms.
• When this is prolonged for some time ( 3-4 days) and if the symptoms do not subside, the doctor would then suggest a Urine Culture and Sensitivity test to determine if there is an infection and to what extent.
• In many cases, if the urine infection levels have increased, the person may also experience high fever and sudden chills in the body. This may go up to 5-6 days as well, if not detected and cured on time.
• Urine culture and sensitivity are used to diagnose, and screen for diseases or medical conditions related to urinary tract infection for persons experiencing symptoms such as frequent and painful urination.

General symptoms of UTIs

1. Painful or difficult urination with a burning sensation
2. Increased urination frequency
3. Urine that smells bad or appears cloudy or reddish
4. Pain and pressure in the lower abdomen (belly) and back
5. Feeling Chills
6. Unexpected fever
7. Malaise
8. Tiredness

Why does the urine culture report takes 3 days?

No all reports take 3 days. Only positive i.e. growth of organism takes 3 days due to following reasons-

• On the first day inoculation of urine on culture media and then incubated for 24 hours.
• If the growth of organisms, on the second day perform biochemical tests as well as AST.
• On 3 days, report for isolation of bacteria/organism and AST pattern.

Urine collection for culture

Collect Mid-Stream Urine (MSU). Clean the genitals before collection, following  these steps to get the sample:

• Initial or the first few drops of the urine should be discarded in the toilet.
• MSU samples should be collected in the sterile container provided.
• The end of the urine should not be collected. Cap the container.
• If delay in test processing, keep it in the refrigerator or a cool place during the collection period.

Test procedure of Urine culture and sensitivity

On the first day

Culture urine  Specimens

Blood agar
Incubate aerobically
MacConkey agar
Incubate aerobically

Or alternate of these two, you can use CLED agar

Day 2 and Onwards

Examine and Report Cultures of Urine  Specimens
Blood agar and MacConkey agar cultures
Look particularly for:
E. coli
Proteus species

Klebsiella species
Enterococcus species
S. saprophyticus  (10-30% of young women)

Pseudomonas
Providentia
Citrobacter
Serratia

Biochemical tests

Depending on the nature of organisms

Antimicrobial susceptibility test

Antimicrobial sensitivity pattern also depends on the nature of organism involvement

On 3rd day

Organism isolated

Antimicrobial sensitivity pattern

Sensitive (S)

Resistant (R)

Intermediate (I)

Result and interpretation

No growth after 24 hours of incubation at 37°C.

or > 10⁵ CFU/ml of   organism (E. coli) isolated

AST pattern

Antibiotic – Sensitive

Antibiotic- Intermediate

Antibiotic-Resistant

Reference Range

>100,000 colonies/ml : Positive

10,000-100,000 colonies/ml: Indeterminate

<10,000 colonies/ml: Negative

Positive means that there is a certain amount of bacteria or organisms that have been found in the Urine Sample and that the infection is certainly because of these bacteria. The Culture report not only detects the various types of germs present but also shows its sensitivity to various Antibiotics. This means you can gauge from this report itself that which antibiotics would provide maximum benefit.

Possible pathogens of urine 

Bacteria
Gram-positive
Staphylococcus saprophyticus
Enterococcus species

Gram-negative

Escherichia coli

Proteus species

Klebsiella species

Pseudonomas aeruginosa

Providentia
Citrobacter
Serratia

Fungi
Candida albicans
Parasites

Trichomonas vaginalis (trophozoite)

Enterobius vermicularis (Ova)

Schistosoma hematobium (ovum)

Commensals
Any commensal organisms found in urine are usually those that have contaminated the specimen from the urethra and they are-
Micrococci
Diphtheroids
Candida species

Mycoplasma species

Mycobacterium smegmatis

Acinetobacter species

Related Videos-

#Pus cells and bacteria under the Microscope of UTI patient urine at a magnification of 400X and slightly higher-

#Pus cells, RBCs, bacteria in the urine of UTI patient: 

History of the patient: A 28 years female with a history of fever, Lower abdomen pain, burning during urination feeling unusual was brought to the hospital of the emergency section after a check-up doctor suggested she check up urine for R/E and blood for complete blood cell count.

Laboratory investigation: blood report: Elevated neutrophils with leukocytosis Urine R/E: Turbid, albumin 4+, sugar-Nil

Microscopy: Pus cells-plenty, RBCs- 6-8, bacteria-4+

The doctor again suggested urine culture and Sensitivity after observing the previous reports of urine R/E and CBC.

Urine C/S Report: Escherichia coli isolated  with more than  10⁵ CFU/mL Sensitivity: Amoxycillin: Resistant

Co-trimoxazole :Resistant

Ofloxacin: Intermediate

Gentamycin: Resistant

Nitrofurantion: Sensitive

After complete medication of nitrofurantoin, the patient gets benefited and recovered from Urinary Tract Infection.

#E coli and pus cells in urine-

#Pus cells,Klebsiella in Urine of Urinary tract infection /UTI patient-

# Experiment No 13.0: Saline/iodine preparation of stool samples for microbiological examination#

Saline Wet Mount for Stool

Saline wet mount for stool or stool wet mount is the simplest and basic method for the study of feces and applicable in every medical laboratory even in small set up. It uses for the following purposes-

1. To observe live trophozoites ( e.g.  Entamoeba histolytica/dispar, Girdia lablia, Trichomonas, etc.) and larvae of parasites ( e.g. Strongyloides stercoralis) are motile except inactive forms.
2. To find out eggs, cysts, oocysts of different parasites ( Helminths, protozoa, and coccidian parasites).
3.  To determine the presence of leukocytes and erythrocytes in a fecal smear.
4. It also gives clues towards the motility of bacteria (Shigella non-motile causing bacillary dysentery whereas Vibrio cholerae shows darting motility which is causative agent cholera).
5. It also remarks the presence of fungal elements ( yeast cells, hyphae, or fungal spores).
6. It also visualizes the presence of non-parasitic structures like Charcot Leyden crystal,  muscle fibers, fat globules, starch cells, vegetable fibers, hair, etc.

Principle of the saline wet mount of stool

Saline wet mount preparation for stool uses analyzing a stool specimen in coprology (study of feces). It utilizes a physiological saline solution (0.85% NaCl ) as an isotonic media to maintain the cellular structure of the various organisms as well as our cells that are found in stool.

Requirements for saline wet mount

• Physiological saline ( 0.85% NaCl)
• Specimen: stool
• Sterile bamboo sticks or low cone on the end of a wooden applicator stick
• Clean and grease-free slides and
• Cove slips(22- by 22-mm)
• Microscope
• Gloves

Saline wet mount of stool Preparation

1. First, wear the groves.
2. Take a clean and grease-free slide.
3. Add one drop of physiological saline and then add a stool equivalent to a match stick head (2 mg)  with the help of a stick.
4. Mix it properly and apply a coverslip over a uniform suspension without creating bubbles.
5. Note: If a fresh stool specimen is received and if blood and mucus are present, the specimen should be examined as a direct mount making sure to sample the bloody areas.
6. Examine the entire 22- by 22-mm coverslip systematically with the low power objective (10X ) and low light intensity.
7.  If any suspicious objects encounter, examine with the high dry objective (40X).

Result Interpretation

Presence of active trophozoite/s: Motile retractile bodies

Cyst, oocyst, egg, inactive trophozite/s, larvae: Retractile bodies and finally focus at high dry power field

Keynotes

1. There is little difference between normal and physiological saline. Physiological saline is  0.85% NaCl whereas normal saline is 0.9% NaCl.
2. Gram’s iodine is not applicable for staining parasitic organisms and for this D’Antoni’s iodine uses.
3. Oil immersion examination is also preferred in parasitology for the permanent stained smear of parasites.
4. Intestinal protozoa can not conform on the basis of a wet mount alone and thus permanent stained smears require to confirm the specific identification of suspected organisms.

Limitations of saline wet mount for stool examination

1. Due to lack of stain, it is difficult to get morphological details.
2. Inappropriate preparation of the smear may hide parasites.
3. Improper adjustment of the microscope in relation to the objective may create problems.

Saline Wet mount Related Videos-

Heavy load of parasites in stool|| Stool microscopic examination|| Trichomonas hominis in a saline wet mount of feces as shown below-

Trichuris trichura or whipworm under saline wet mount at 40X objective under the microscope –
Features: Barrel shape
Mucus thread at each pole as shown in the video

Egg of Taenia species
They are spherical, brown in color (bile stained), and measure 30-40 µm in diameter.
They are surrounded by embryophore which is brown, thick-walled, and radially striated.
Inside embryophore, the hexacanth embryo (oncosphere) presents three pairs of hooklets.
They do not float in a saturated solution of common salt (brine solution).
They are viable for 8 weeks.

Roundworm or Ascaris lumbicoides egg under the microscope in saline preparation
egg-infertile
Finding of eggs
In stool:
direct microscopic examination of a saline emulsion of the stool
Concentration methods may be used.
Note:
The fertilized egg floats in salt solution
Unfertilized eggs do not float

Hymenolypsis (now called Vampirolepsis)  egg in a saline wet mount of stool: showing hooklets egg of Hymenolepis liberated in feces by the gradual disintegration of terminal segments

Spherical or oval in shape, 30-45 µm

Two distinct membranes

Outer membranes are thin and colorless

Inner embryophore encloses an oncosphere with 3 pairs of hooklets

Space between two membranes –filled with yolk granules and polar filaments emanating from little knobs at either end of embryophore.

Egg of hookworm ( Ancylostoma duodenale or Necator americanus)  in saline preparation of stool under the microscope
Egg features-
Shape: oval or elliptical with flattened poles( one pole more often flattened than other), size: 65 X 40 um, color: colorless ( no bile stain), dark brown as stained with iodine. Shell: very thin transparent hyaline shell membrane, appears as a black line and contains: segmented ovum with 4 blastomeres, has a clear space between eggshell and segmented ovum. Float in saturated NaCl. Type: A( fresh stool) : 4 ,8, 16 grey granular cell clear blastomeres. Type: B( few hours old): a uniform mass of many grey granular cells. Type: C( 12-48 hr): the whole egg is filled with larva, embryonated.

Enterobious vermicularis ( common manes pinworm or threadworm or seatworm) – eggs and some are with larva in a saline wet mount of feces –
Shape: oval, planoconvex.

Size : 50-60μm x 20-30μm.

Surrounded by double-layered eggshell

Embryonated when passed fresh; contains a tadpole larva inside.

Entamoeba coli Cyst with 8 nuclei in iodine wet mount as shown below-

Giardia lamblia cysts in iodine wet mount as shown below ( look at center)-

Entamoeba histolytica (Amoeba) trophozoites and cyst in LPCB preparation as shown below-

Blastocystis hominis cyst in Sargeant stained slide under the microscope as shown below-

Oocyst of Cyclospora cayetanensis ( coccidian parasite) in a saline wet mount of stool under the microscope as shown below-

# Experiment No 14.0:  Microbiological examination / biochemical tests of vaginal swabs#

Introduction of Vaginal Wet Mount

The vaginal wet mount also called the vaginitis test is very useful for diagnosis vaginal infections that could be causing vaginitis. Vaginitis encompasses a variety of disorders that cause infection or inflammation of the vagina. The causative agents of vaginitis can include bacteria, yeast, viruses, and parasites. Non-infectious vaginitis may also occur due to estrogen imbalance (low level)  that causes vaginal dryness as well as a reaction to vaginal products such as soap, bath oils, spermicidal jelly, or douches.

The most common pathogens are as follows-

Bacteria: Gardnarella is a commonly found bacteria in the vagina. Overgrowth results in bacterial vaginosis, Streptococcus or Staphylococcus can also be present but usually do not result in an infection. A bacterial infection can cause a grayish-white discharge with a fishy odor.

Fungi: Candida albicans cause genital itching and a thick, white vaginal discharge with a cottage cheese-like texture.
Viruses:  Human papillomavirus (HPV)
Parasites: Trichomonas vaginalis, Pinworms, scabies, and lice can cause inflammation of the vulva and vagina.
Vaginitis is also a sexually transmitted infection (STI).

Symptoms of vaginitis

Common symptoms of vaginitis are as follows-

1. vaginal discharge that may have an odor
2. itching or swelling on the outside of the vagina
3. burning during urination
4. pain or discomfort during intercourse

Principle of wet mount

Saline wet mount preparation for vaginal smear analyzing a vaginal discharge specimen for diagnosis the causative agents of vaginitis. It utilizes a physiological saline solution (0.85% NaCl ) as an isotonic media to maintain the cellular structure of the various organisms as well as patient cells that are found in vaginal discharge.

Vaginal Wet Mount  or Vaginal smear Preparation

A wet mount of vaginal smear also called wet preparation uses to find out the causative agents of vaginal infections that don’t affect the urinary tract. A clinician ( gynecologist) will have you lie down on an exam table with your feet in stirrups, like at a regular gynecologic exam. She/he will insert a speculum into the vagina to help see the area. After that, a sterile cotton swab is inserted into the vagina to obtain a sample of vaginal discharge. The clinician will transfer the sample onto a slide or leaving on the swab. The slide is sent to the microbiology section to check the causative agent of vaginitis.

Requirements for the vaginal wet mount test procedure

• Test slides or vaginal discharge on the swab
• Physiological saline or normal saline
• Microscope
• Coverslips
• Sterile Wooden Applicator Stick
• Gloves

Wet mount procedure

1. First, wear gloves.
2. Take a clean and grease-free slide ( no need in case of slides provided by a clinician).
3. Place s sample of the vaginal discharge on a glass slide.
4. Add one drop of physiological or normal saline
5. Mix it properly with a sterile wooden applicator stick and apply a coverslip over a uniform suspension without creating bubbles.
6. Examine the entire 22- by 22-mm coverslip systematically with the low power objective (10X ) and low light intensity.
7.  If any suspicious objects encounter, examine with the high dry objective (40X).

Observation of Vaginal smear

Look at under a microscope for bacteria, yeast cells, Trichomonas, pus cells ( dead white blood cell), and clue cells that are an indicator of bacterial vaginosis.

Result Interpretation of  vaginal smear or vaginal  wet mount 

Bacteria:  The organisms which have a size of this range 0.2 – 2.0 × 1 – 10 µm and shape of normally round or rod.

Trichomonas vaginalis:  A moving object usually the size of 10 μm in length and 7 μm in width.

Yeast cells: Oval or round in shape having a size of usually 3-5µm  and some with budding

Pus cells:  Dead white blood cells form pus and the pus cells having a size of usually  12 to 14 µm with lobes.

Clue cells: Clue cells are epithelial cells of the vagina that get their distinctive stippled appearance by being covered with bacteria.

Uses of wet mount or vaginal smear

It is very useful in vaginal infections to find out –

• Bacteria
• Yeast cells
• Trichomonas
• Pus cells and
• A clue cell is an indicator of bacterial vaginosis.
• vaginal pH determination-The normal vaginal pH is 3.8 to 4.5. Bacterial vaginosis, trichomoniasis, and atrophic vaginitis often cause a vaginal pH higher than 4.5.
• Whiff test. Several drops of a potassium hydroxide (KOH) solution are added to a sample of the vaginal discharge. A strong fishy odor from the mix means bacterial vaginosis is present.
• KOH mount: A sample of the vaginal discharge is placed on a slide and mixed with a solution of potassium hydroxide (KOH). The KOH kills bacteria and cells from the vagina, leaving only yeast for a yeast infection.
• It may also be used in a rape investigation to detect the presence of sperm.

Limitation of wet mount or vaginal smear

1. Infections like chlamydia, genital warts, syphilis, herpes simplex, and gonorrhea can also affect the vagina, but the causative agents of these diseases can not be covered by this wet mount.
2. Due to the lack of stain, it is difficult to get morphological details.
3. Inappropriate preparation of the smear may hide parasites.
4. Improper adjustment of the microscope in relation to the objective may create problems.

Key Notes

1. A properly prepared wet mount should be thin enough to allow newsprint to be visible.
2. There is little difference between normal and physiological saline. Physiological saline is  0.85% NaCl whereas normal saline is 0.9% NaCl.
3. Oil immersion examination is also preferred in parasitology for the permanent stained smear of parasites.
4. Intestinal protozoa can not conform on the basis of a wet mount alone and thus permanent stained smears require to confirm the specific identification of suspected organisms.

Related videos of Vaginal smears

# Vaginal swab Wet preparation under Microscope ||Bacteria ||RBCs ||Pus cells || Epithelial cells as shown below-

#Trichomonas, pus cells, epithelial cells, RBCs in vaginal swab microscopy

#Clue cells under the Microscope as shown below-

#Experiment No 15.0: Demonstration of VDRL/RPR test, WIDAL test#

Rapid Plasma Reagin (RPR) Test

Card demonstrating RPR Test-
It works on the principle of flocculation and flocculation is a special type of precipitation where precipitates remain suspended instead of sedimentation.
PC-Reactive

NC-Non-reactive

Principle

In the RPR test, the RPR antigen is mixed with unheated or heated serum or with unheated plasma on a plastic-coated card. The reagin binds to the test antigen which consists of cardiolipin-lecithin coated particles that cause macroscopic flocculation resulting in aggregation of carbon particles. The flocculation appears as black clumps against the white background of the plastic-coated card. The RPR test measures IgM and IgG antibodies to lipoidal material released from damaged host cells as well as to lipoprotein-like material, and possibly cardiolipin released from the treponemes. Antilipoidal antibodies are antibodies that are produced not only as a consequence of syphilis and other treponemal diseases but also in response to nontreponemal diseases of an acute and chronic nature in which tissue damage occurs. The antigen is prepared from a modified Venereal Disease Research Laboratory (VDRL) antigen suspension containing choline chloride to eliminate the need to heat inactivate serum, ethylenediaminetetraacetic acid (EDTA) to enhance the stability of the suspension and finely divided charcoal particles as a visualizing agent.

Test Specimens

Serum /Plasma

Test Reagents

RPR antigen suspension:– stabilized combination of 0.003% cardiolipin, 0.020-0.022% lecithin, 0.09% cholesterol, 10% choline chloride, 0.0125M EDTA, 0.01875% charcoal, 0.01M Na2HP04, 0.01M KH2P04, 0.1% thimerosal in distilled water.

Control serum samples:- Control serum samples are lyophilized reactive (R), minimally reactive (Rm), and nonreactive (N) control serum specimens.

0.9% Saline:- Add 0.9 g of dry sodium chloride (ACS) to 100 ml of distilled water. Disposable, calibrated 20-gauge needle without bevel.

Plastic antigen dispensing bottle

Plastic-coated RPR cards, with 10 circles, each approximately 18 mm in diameter.

Mechanical rotator, fixed-speed or adjustable to 100 + 2 rpm

High-intensity incandescent lamp

Safety pipetting device with a disposable tip that delivers 50 μl

Procedure of RPR test

RPR Qualitative Test

Place 50 μl of serum or plasma onto an 18-mm circle of the RPR test card. Using the inverted Dispenstir (closed-end) or flat toothpicks, spread the serum or plasma to fill the entire circle. Do not spread the specimen beyond the confines of the circle. Gently shake the antigen dispensing bottle to resuspend the particles. Holding the dispensing bottle and needle in a vertical position, exactly 1 free-falling drop (17 μl) of antigen suspension to each circle containing serum or plasma. Do not mix. Place the card on the mechanical rotator under a humidifying cover. Rotate the card for 8 minutes at 100 + 2 rpm. Immediately remove the card from the rotator; briefly rotate and tilt the card by hand (three or four to-and-fro motions) to aid in differentiating nonreactive from minimally reactive results)

Reading and Reporting Qualitative RPR test Results

Read the test reactions under a high-intensity incandescent lamp. Reading Report
Characteristic clumping ranging from marked and intense (reactive) to slight but definite (minimally to moderately reactive)- Reactive (R)
Slight roughness or no clumping -Nonreactive (N)

Note: Only two reports with the RPR card test are possible: reactive, no matter how much clumping, or nonreactive.

RPR Quantitative Test

Dilute to an endpoint titer all serum specimens with rough nonreactive results in the qualitative test. Test each specimen undiluted (1:1), and in 1:2, 1:4, 1:8, and 1:16 dilutions. Place 50 μl of 0.9% saline in circles numbered 2 through 5. Do not spread the saline. Using a safety pipette device, place 50 μl of serum in circle 1 and 50 μl of serum in circle 2. Mix the saline and the serum in circle 2 by drawing the mixture up and down in a safety pipette eight times. Transfer 50 μl from circle 2 (1:2) to circle 3, and mix.Transfer 50 μl from circle 3 (1:4) to circle 4, and mix. Transfer 50 μl from circle 4 (1:8) to circle 5 (1:16), mix, and then discard the last 50 μl. Proceed as RPR qualitative test. If reactive at last dilution further dilute the serum and test.After completing the tests, remove the needle from the antigen dispensing bottle. Rinse the needle in distilled water, and air dry. Reading and Reporting quantitative results
Read the test reaction under a high-intensity incandescent lamp as for the qualitative test.

Result Interpretation

A reactive RPR card test may suggest past or present infection with a pathogenic treponeme; however, it may also be a false-positive reaction. A nonreactive RPR card test without clinical evidence of syphilis may suggest no current infection or an effectively treated infection. A nonreactive RPR card test with clinical evidence of syphilis can be seen in early primary syphilis; in secondary syphilis, as a result of the prozone reaction; and in some cases of late syphilis. A nonreactive RPR card test result does not rule out an incubating syphilis infection. When the quantitative RPR card test is performed on patients with syphilis, a fourfold rise in titer in a repeat specimen may suggest an infection, reinfection, or a treatment failure, a fourfold decrease, e.g. 1:16 to 1:4, in titer following treatment for early syphilis usually indicates that therapy was adequate. All reactive qualitative RPR card tests should be diluted to an endpoint and the endpoint titer reported. Unusually high RPR card test titers can be seen with concurrent human immunodeficiency virus type 1 (HIV-1) infection. Unusually high false-positive titers may also be seen in patients with lymphomas.

Limitations of RPR test

A prozone reaction may be encountered occasionally. The RPR card test may be reactive in persons from areas where yaws, pinta, or nonvenereal syphilis is endemic. Biological false-positive (BFP) reactions occur occasionally. Non-syphilitic conditions giving biologic false-positive results in non-treponemal tests like malaria, leprosy, relapsing fever, infectious mononucleosis, atypical pneumonia, infectious hepatitis, rheumatoid arthritis, pregnancy, aging individuals, viral pneumonia, lupus erythematous, measles, pregnancy, drug abuse, pneumococcal pneumonia, etc.

False Negative Reactions

1. Technical error – unsatisfactory antigen or technique
2. Low antibody titers
3. Presence of inhibitors in the patient’s serum
4. Reduced ambient temperature (below 23°C to 29°C)
5. Prozone reaction

Note: Venereal Disease Research Laboratory (VDRL) Versus Rapid Plasma Reagin test:

Why VDRL is replaced by the RPR test?

Because of the following features-

1. In the RPR test- There is no need of preparing fresh reagents daily.
2. No need for a microscope
3. No need for heat inactivation of serum sample
4. But one demerit of the RPR test- the CSF sample can not be tested.

#VDRL Versus RPR test-

#Experiment No. 16: Microbiological examination, biochemical tests of pus sample from wound#

Introduction of Pus, Ulcer Material, Skin Specimens

Pus, Ulcer Material, Skin Specimens are common samples for microbiological examination in a tertiary care center set up like gram stain, culture, and sensitivity.

Sample collection

Abscess

Container: Aerobic swab moistened with Stuart’s or Amie’s medium

Patient preparation: Wipe the area with sterile saline or 70% alcohol. Swab along the leading edge of the wound.

Transport to the laboratory: Within 24 hours art room temperature.

Attention

Pus from an abscess is best collected at the time the abscess is incised and drained, or after it has ruptured naturally. When collecting pus from abscesses, wounds, or other sites, special care should be taken to avoid contaminating the specimen with commensal organisms from the skin. As far as possible, a specimen from a wound should be collected before an antiseptic dressing is applied.

Describe Specimen

For additional investigations
Look for granules: When mycetoma or actinomycosis is
suspected

Culture Pus, Ulcer Material, Skin Specimens

Blood agar
Incubate aerobically
MacConkey agar
Incubate aerobically
Cooked meat medium

For anaerobic bacteria
Subculture at 24 hours, 48 hours, and 72 hours as indicated

Optional
Neomycin blood agar when
anaerobic infection is suspected
Incubate anaerobically up to 48 h
Culture for M. tuberculosis or M. ulcerans
Requires facilities of a
Tuberculosis Reference Laboratory

Examine Microscopically of Pus, Ulcer Material, Skin Specimens

Gram smear
For pus cells and bacteria

Optional steps 
Ziehl-Neelsen smear: When tuberculosis or M. ulcerans disease is suspected
KOH preparation:
When a fungal or actinomycete infection is suspected
Giemsa or Wayson’s smear:
When bubonic plague is suspected
Polychrome methylene blue:
When cutaneous anthrax is
suspected
Dark-field microscopy:
To detect treponemes when
yaws or pinta is suspected

Day 2 and Onwards

Examine and Report Cultures of Pus, Ulcer Material, Skin Specimens
Blood agar and MacConkey agar cultures
Look particularly for:
S. aureus
S. pyogenes
P. aeruginosa
Proteus species
E. coli
Enterococcus species
Klebsiella species
Anaerobes:
C. perfringens
Bacteroides fragilis group
Peptostreptococcus species

Biochemical tests

Depending on the nature of organisms

Antimicrobial susceptibility test

Antimicrobial sensitivity pattern also depends on the nature of organism involvement

On 3rd day

Organism isolated

Antimicrobial sensitivity pattern

Sensitive (S)

Resistant (R)

Intermediate (I)

Possible pathogens of pus

Bacteria
Gram-positive
Staphylococcus aureus
Streptococcus pyogenes
Enterococcus species
Anaerobic streptococci
Other streptococci
Clostridium perfringens
and other clostridia
Actinomycetes
Actinomyces israeli
Also Mycobacterium tuberculosis
Gram-negative
Pseudonomas aeruginosa
Proteus species
Escherichia coli
Bacteriodes species
Klebsiella species
Pasteurella species
Fungi
Histoplasma
Candida albicans,
mycetoma causing fungi
Parasites
Entamoeba histolytica
(in pus aspirated from an amoebic liver abscess)

Commensals
Any commensal organisms found in pus are usually those that have contaminated the specimen from skin, clothing, soil, or from the air if an open wound.

Ulcer Material and Skin Specimens 
Possible pathogens
Bacteria
Gram-positive
Staphylococcus aureus
Streptococcus pyogenes
Enterococcus species
Anaerobic streptococci
Erysipelothrix rhusiopathiae
Bacillus anthracis
Gram-negative
Escherichia coli
Proteus
Pseudomonas aeruginosa
Yersinia pestis
Vincent’s organisms
Mycobacterium leprae

Mycobacterium ulcerans,

Treponema carateum,

 Treponema pertenue.
Viruses
Poxviruses

herpes viruses
Fungi
Dermatophytes (ringworm fungi)
Malassezia furfur
Fungi that cause chromoblastomycosis
Candida albicans

Parasites
Leishmania species
Onchocerca volvulus
Dracunculus medinensis

Commensals
Commensal organisms that may be found on the
skin include:
Gram-positive
Staphylococci
Micrococci
Anaerobic cocci
Viridans streptococci
Enterococci
Diphtheroids
Propionibacterium acnes
Gram-negative
Escherichia coli
and other coliforms

#Experiment No. 17: Microscopical examination of ova of common intestinal parasites#

Saline Wet Mount for Stool

Saline wet mount for stool or stool wet mount is the simplest and basic method for the study of feces and applicable in every medical laboratory even in small set up. It uses for the following purposes-

1. To observe live trophozoites ( e.g.  Entamoeba histolytica/dispar, Girdia lablia, Trichomonas, etc.) and larvae of parasites ( e.g. Strongyloides stercoralis) are motile except inactive forms.
2. To find out eggs, cysts, oocysts of different parasites ( Helminths, protozoa, and coccidian parasites).
3.  To determine the presence of leukocytes and erythrocytes in a fecal smear.
4. It also gives clues towards the motility of bacteria (Shigella non-motile causing bacillary dysentery whereas Vibrio cholerae shows darting motility which is causative agent cholera).
5. It also remarks the presence of fungal elements ( yeast cells, hyphae, or fungal spores).
6. It also visualizes the presence of non-parasitic structures like Charcot Leyden crystal,  muscle fibers, fat globules, starch cells, vegetable fibers, hair, etc.

Principle of the saline wet mount of stool

Saline wet mount preparation for stool uses analyzing a stool specimen in coprology (study of feces). It utilizes a physiological saline solution (0.85% NaCl ) as an isotonic media to maintain the cellular structure of the various organisms as well as our cells that are found in stool.

Requirements for saline wet mount

• Physiological saline ( 0.85% NaCl)
• Specimen: stool
• Sterile bamboo sticks or low cone on the end of a wooden applicator stick
• Clean and grease-free slides and
• Cove slips(22- by 22-mm)
• Microscope
• Gloves

Saline wet mount of stool Preparation

1. First, wear the groves.
2. Take a clean and grease-free slide.
3. Add one drop of physiological saline and then add a stool equivalent to a match stick head (2 mg)  with the help of a stick.
4. Mix it properly and apply a coverslip over a uniform suspension without creating bubbles.
5. Note: If a fresh stool specimen is received and if blood and mucus are present, the specimen should be examined as a direct mount making sure to sample the bloody areas.
6. Examine the entire 22- by 22-mm coverslip systematically with the low power objective (10X ) and low light intensity.
7.  If any suspicious objects encounter, examine with the high dry objective (40X).

Result Interpretation

Presence of active trophozoite/s: Motile retractile bodies

Cyst, oocyst, egg, inactive trophozite/s, larvae: Retractile bodies and finally focus at high dry power field

Keynotes

1. There is little difference between normal and physiological saline. Physiological saline is  0.85% NaCl whereas normal saline is 0.9% NaCl.
2. Gram’s iodine is not applicable for staining parasitic organisms and for this D’Antoni’s iodine uses.
3. Oil immersion examination is also preferred in parasitology for the permanent stained smear of parasites.
4. Intestinal protozoa can not conform on the basis of a wet mount alone and thus permanent stained smears require to confirm the specific identification of suspected organisms.

Limitations of saline wet mount for stool examination

1. Due to the lack of stain, it is difficult to get morphological details.
2. Inappropriate preparation of the smear may hide parasites.
3. Improper adjustment of the microscope in relation to the objective may create problems.

Saline Wet mount Related Videos-

Heavy load of parasites in stool|| Stool microscopic examination|| Trichomonas hominis in a saline wet mount of feces as shown below-

Trichuris trichura or whipworm under saline wet mount at 40X objective under the microscope –
Features: Barrel shape
Mucus thread at each pole as shown in the video

Egg of Taenia species
They are spherical, brown in color (bile stained), and measure 30-40 µm in diameter.
They are surrounded by embryophore which is brown, thick-walled, and radially striated.
Inside embryophore, the hexacanth embryo (oncosphere) presents three pairs of hooklets.
They do not float in a saturated solution of common salt (brine solution).
They are viable for 8 weeks.

Roundworm or Ascaris lumbicoides egg under the microscope in saline preparation
egg-infertile
Finding of eggs
In stool:
direct microscopic examination of a saline emulsion of the stool
Concentration methods may be used.
Note:
The fertilized egg floats in salt solution
Unfertilized eggs do not float

Hymenolypsis (now called Vampirolepsis)  egg in a saline wet mount of stool: showing hooklets egg of Hymenolepis liberated in feces by the gradual disintegration of terminal segments

Spherical or oval in shape, 30-45 µm

Two distinct membranes

Outer membranes are thin and colorless

Inner embryophore encloses an oncosphere with 3 pairs of hooklets

Space between two membranes –filled with yolk granules and polar filaments emanating from little knobs at either end of embryophore.

Egg of hookworm ( Ancylostoma duodenale or Necator americanus)  in saline preparation of stool under the microscope
Egg features-
Shape: oval or elliptical with flattened poles( one pole more often flattened than other), size: 65 X 40 um, color: colorless ( no bile stain), dark brown as stained with iodine. Shell: very thin transparent hyaline shell membrane, appears as a black line and contains: segmented ovum with 4 blastomeres, has a clear space between eggshell and segmented ovum. Float in saturated NaCl. Type: A( fresh stool) : 4 ,8, 16 grey granular cell clear blastomeres. Type: B(a few hours old): a uniform mass of many grey granular cells. Type: C( 12-48 hr): the whole egg is filled with larva, embryonated.

Enterobious vermicularis ( common manes pinworm or threadworm or seatworm) – eggs and some are with larva in a saline wet mount of feces –
Shape: oval, planoconvex.

Size : 50-60μm x 20-30μm.

Surrounded by double layered eggshell

Embryonated when passed fresh; contains a tadpole larva inside.

Entamoeba coli Cyst with 8 nuclei in iodine wet mount as shown below-

Giardia lamblia cysts in iodine wet mount as shown below ( look at center)-

Entamoeba histolytica (Amoeba) trophozoites and cyst in LPCB preparation as shown below-

Blastocystis hominis cyst in Sargeaunt stained slide under the microscope as shown below-

Oocyst of Cyclospora cayetanensis ( coccidian parasite) in a saline wet mount of stool under the microscope as shown below-

#Experiment No 18: Study of blood slides for parasite examination#

Blood parasites are also called haemoparasites are those parasites that live within their host bloodstream.

The parasites which are found in blood are –

1. Malarial parasite
2. Filaria
3. Leishmania
4. Trypanosoma
5. Babesia

Malarial parasite: It is a protozoan disease transmitted by the bite of an infected female anopheles mosquito. There are four species of Plasmodium and they are- P. vivax, P. falciparum,  P. malariae and P. ovale. Parasitic structures, rings, trophozoites, schizont, and gametocytes may be found on smears of blood.

Grading of Parasitic Load ( Plasmodium)

1 – 10 Parasites /100 fields: +

11 – 100 Parasites /100 fields:  + +

1 – 10 Parasites/field:  + + +

>10 Parasites /Field:  + + + +

Filaria

Filariasis is an infectious disease caused by nematodes, filaria. e.g. Wuchereria bancrofti, Brugia malayi,  Oncocerca volvulus and Loa loa. These are spread by blood-feeding insects such as black flies and mosquitoes. The filarial worms reside in the subcutaneous tissues, lymphatic system, or body cavities of humans,

Classification of filarial worms  based on location in the body 

1. lymphatic filariasis – causative agents: Wuchereria bancrofti, Brugia malayi, Brugia timorii, 
2. Subcutaneous filariasis: Causative agents are-Loa loa, Oncocerca volvulus, Mansonella streptocerca
3. Serous cavity filariasis: Causative agents are-Mansonella perstans, Mansonella ozzardi (They are virtually non-pathogenic)

Microfilaria

The females of parasites are viviparously giving birth to larvae so-called microfilaria. This parasite includes four genera and species that are parasitic to humans. These filarial nematodes are as follows: 1. Wuchereria bancrofti 2. Brugia malayi 3. Oncocerca volvulus and 4. Loa loabut mainly two genera i.e. Wuchereria and Brugia are encountered in peripheral blood.

Wuchereria bancrofti

Habitat: Lymphatics and lymph nodes

Morphology

It has two stages adult worm and larva (microfilaria).

Adult worm

The adults are whitish, translucent, thread-like worms with smooth cuticles and a tapering ends. The female is larger (70–100 × 0.25 mm) than the male (25–40 × 0.1 mm). The posterior end of the female worm is straight, while that of the male is curved vertically and contains 2 spicules of unequal length. Males and females remain coiled together usually in the abdominal and inguinal lymphatics and in the testicular tissues. The female worm is viviparous and directly liberates sheathed microfilariae into the lymph.

Lifespan: 10 to 15 years

Microfilaria

The microfilaria has a colorless, translucent body with a blunt head, and pointed tail It measures 250–300 µm in length and 6–10 µm in thickness
It is covered by a hyaline sheath, within which it can actively move forwards and backward Along the central axis of the microfilaria, a column of granules can be seen, which are called somatic cells or nuclei. The granules are absent at certain specific locations—a feature that helps in the identification of the species. Microfilariae do not multiply or undergo any further development in the human body. Their lifespan is believed to be about 2–3 months. It is estimated that a micro filarial density of at least 15 per drop of blood is necessary for infecting mosquitoes

Note:  Microfilaria is differentiated on sheath pattern, nuclei distribution, and size.

Periodicity

The microfilariae circulate in the bloodstream. They show a nocturnal periodicity in peripheral circulation; being seen in large numbers in peripheral blood only at night (between 10 pm and 4 am). This correlates with the night-biting habit of the vector mosquito.

Leishmania

 Introduction of Leishmaniasis

Leishmaniasis is an infectious disease caused by the unicellular blood parasite, Leishmania. Members of the genus Leishmania pass their life cycle in two hosts- man and the insect vector female sandfly. On the basis of clinical disease production, it is of various types like Visceral leishmaniasis, Cutaneous leishmaniasis, and cutaneous and mucocutaneous leishmaniasis. So, here we discuss visceral leishmaniasis in detail and it is also called Kala-azar, which is the most severe form of Leishmaniasis.

Classification of Leishmaniasis

Classification of Leishmaniasis ( based on disease production )

• Visceral leishmaniasis
  Leishmania donovani (complex) (VL): L. donovani, L. Infantum, and L. chagas
•  Cutaneous leishmaniasis of the Old world:

Leishmania tropica
Leishmania major

• Cutaneous and mucocutaneous leishmaniasis of New world
  Leishmania mexicana (Complex)
  Leishmania brazilliensis (complex)
  Leishmania peruriana

Leishmania donovani

It causes visceral leishmaniasis or kala-azar and also causes post-kala-azar dermal leishmaniasis.

Habitat:  Amastgote form in the reticuloendothelial system (macrophages): e.g. spleen, bone marrow, liver, etc.
Transmission: Persons to person transmitted by the bite of female sand flies (Phlebotomus or Lutzomyia).

Morphology of Leishmania donovani

It has two forms promastigote and amastigote.

Promastigote
It is long slender, spindle-shaped about 15-20 µm length, 1-2µm width.

The nucleus is centrally placed.

Kinetoplast (blepharoplasty and parabasal body ) lies transversely and near the anterior end.

Flagellum projects from the anterior end and may be of the size of the body or even longer.
Habitat: a digestive tract of insect vector (sandfly) or laboratory culture.

Amastigote
Amastigote form is also called LD bodies.
It has shape and size: round or oval body, 2-4 micrometer along the longitudinal axis.

Kinetoplast  (parabasal body and the blepharoplast ) at a right angle to the nucleus.

Axoneme arises from blepharoplast and extends up to the tip of the cell.

Vacuole alongside the axoneme is present.
Habitat: It is present in reticulo-endothelial cells of the body.

Grading of parasitic Load ( Leishmania)

•  >100 Parasites/Field:  6+
• 10 -100 Parasites/Field:  5+
•  1 -10 Parasites/ Field:  4+
• 1 – 10 Parasites/10 Fields: 3+
•  1 -10 Parasites/100 Fields:  2+
• 1- 10 Parasites/1000 Fields:  1+
•  O Parasites/1000 Fields: 0

Trypanosoma

They are haemoflagellates that live in the blood and tissue of their hosts. They cause two important diseases.

1. Sleeping diseases: It is also called African trypanosomiasis and is caused by Trypanosoma brucei.
2. Chagas diseases: It is also called American trypanosomiasis, and  is caused by protozoan parasites, Trypanosoma cruzi.

Laboratory Diagnosis of Trypanosoma

• Wet, thin, and thick blood film
• Concentration method
• Serodiagnosis -IHA , IFA, ELISA
• Molecular diagnosis
• Blood culture- Novy-MacNeal-Nicolle medium (NNN)  and Liver infusion tryptose (LIT) medium
• Animal inoculation (mice)

Babesia

Babesiosis is caused by microscopic parasites, Babesia that infects red blood cells and is spread by ticks(Ixodes scapularis).  3 species of Babesia are –

B. bovis ,

B. divergens ,

B. microti.

 Habitat: Inside RBCs

Morphology of Babesia

Infective form

sporozoite (ticks)

Trophozoites

• Oval and spindle-shaped
•  size 1 – 5 µm
• Small chromatin dot and scanty cytoplasm

Laboratory Diagnosis of Babesia

1. A thin and  thick smear
2. Stain-Leishman, Wrights, Giemsa
3.  Only ring form seen

 Features

• Haemozoin and  gametocyte are absent
•  Merozoite arranged in tetrads or maltese cross form

Further Readings

1.  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
2.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
3. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
4. Clinical Microbiology Procedure Handbook Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
5. https://www.researchgate.net/publication/331899509_Hot_Air_Oven_for_Sterilization_Definition_Working_Principle
6. http://www.acmasindia.com/blog/hot-air-oven/
7. https://ultrasoniccleanersmfgr.com/hot-air-oven.html  gclid=CjwKCAjwps
8. https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3340325
9. https://www.stericox.com/laboratory-oven/hot-air-oven.html
10. https://www.prestogroup.com/blog/use-of-hot-air-oven-in-laboratories/
11. https://en.wikipedia.org/wiki/Hot_air_oven
12. https://www.sciencedirect.com/topics/engineering/autoclave
13. https://en.wikipedia.org/wiki/Autoclavehttps://tuttnauer.com/blog/autoclave
14. https://www.steris.com/healthcare/knowledge-center/sterile-processing/everything-about-autoclaves
15. https://ehs.princeton.edu/book/export/html/380
16. https://www.cdc.gov/infectioncontrol/guidelines/disinfection/sterilization/steam.html
17. https://www.explainthatstuff.com/autoclaves.html
18. https://www.britannica.com/science/Bunsen-burner
19. https://www.sciencedirect.com/topics/engineering/bunsen-burner
20. https://www.goodscience.com.au/year-7-science/the-bunsen
21. https://en.wikipedia.org/wiki/Bunsen_burner
22. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
23. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC180726/
24. https://www.asmscience.org/content/education/protocol/protocol.2886
25. https://www.sigmaaldrich.com/catalog/product/sigma/ht90a?lang=en
26. Koneman Color Atlas and Text-Book of Diagnostic -Microbiology-6th  Edn.
27. Jawetz Melnick and Adelberg’s Medical Microbiology-25th Edn.
28. Lippincott’s –Illustrated- review-Microbiology-3rd Edn.
29. Mandell’s Infectious Disease-7th Edn.
30. https://www.sciencedirect.com/topics/medicine-and-dentistry/vagina-smear
31. https://www.uofmhealth.org/health-library/hw6026#hw6029
32. https://www.healthline.com/health/vaginitis-test-wet-mount
33. https://en.wikipedia.org/wiki/High_vaginal_swab
34. https://en.wikipedia.org/wiki/Vaginal_wet_mounthttps://en.wikipedia.org/wiki/Clue_cell
35. https://www.merriam-webster.com/medical/vaginal%20smear
36. https://www.immunology.org/public-information/bitesized-immunology/experimental-techniques/enzyme-linked-immunosorbent-assay
37. https://www.who.int/diagnostics_laboratory/faq/elisa/en/
38. https://www.ncbi.nlm.nih.gov/books/NBK555922/
39. http://www.biobest.co.uk/diagnostics/techniques/elisa-how-does-the-test-work.html
40. https://stanfordhealthcare.org/medical-conditions/sexual-and-reproductive-health/hiv-aids/diagnosis/elisa.html
41. https://www.sciencedirect.com/topics/immunology-and-microbiology/elisa
42. Textbook of Medical Laboratory Technology by Praful B. Godkar, Darshan P. Godkar
43. Textbook of Medical Laboratory Technology by Ramnik Sood (2006)