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Microbiology Short Notes

The topic ‘ Microbiology Short Notes’  will serve as a capsule for candidates who are participating posts for medical lab technicians,  medical lab technologist,s and such related fields.

1. Father of microbiology →Antony van Leeuwenhoek
2. Father of modern microbiology → Louis Pasteur
3. Father of medical microbiology → Robert Koch
4. Father of modern bacteriology→ Robert Koch
5. Father of medicine → Hippocrates
6. Father of virology   →Ivanowsky
7. Father of Antiseptic surgery  → Joseph Lister
8. Father of blood group  → Karl Landsteiner

Milestones in microbiology

Antony van Leeuwenhoek

→ Developed 1 st microscope and observed bacteria for 1 st time.

Louis Pasteur

A French chemist knew as the father of modern microbiology

• Establish Germ theory of disease
• Establish technique of immunization
• Discovered rabies vaccine
• Discovered pasteurization process.
• Introduce sterilization technique

Robert Koch

• Father of medical microbiology
• Father of modern bacteriology
• Formulated Koch’s postulates
• Developed pure culture technique
•  the discovered causative agent of tuberculosis

Joseph Lister

• Father of antiseptic surgery.

Ronald Ross

• Discovered cause of transmission of malaria
• Laveram – Discovered  Malaria Parasite

Characteristic of a prokaryotic organism

1. Nuclear membrane, nucleolus, and cytoplasmic organelles are absent.
2. Single chromosome present
3. The ribosome is 70 s type.
4. Multiplication by binary fission
5. Respiration through mesosomes

General Bacteriology

• The study of bacteria is bacteriology.
• Size  → 0.2 to 1.5 μm in diameter 2-10 μm in length.
• The nuclear material of bacteria is in the form of the nucleoid, which consists of both DNA  and RNA.
• Mycoplasma, spheroplast, and L. forms are bacteria lacking cell walls.

Classification

1. Based on the required temperature

• Thermophilic →Optimum temperature for growth is between 60 to 80 ° C e.g. Bacillus stearothermophilus.
• Mesophilic → Majority of pathogenic bacteria (37 °C to 40°C)
• Psychrophilic → Those which can grow below  20 °C, can grow as low as 2 to 6 ° C.

2. Based on morphology

• Coccobacilli →  Bordetella pertussis , Haenophilus
• Spherical shaped →  Cocci e.g. Staphylococci
• Rod-shaped →  bacilli e.g. E. coli, Salmonella, Klebsiella
• Spiral shaped →  e.g. Campylobacter, Helicobacter
• Comma shaped →  e.g. Vibrio
• Spirochetes →  Treponema, Leptospira , Borellia

Cocci are classified into-

• Micrococci: Only a single spherical cell represents bacterium e.g. Micoccous agilis
• Diplococci: Cocci divide  into one plane and remains in pair e.g. Meaningococcus, gonococcus, pneumococci
• Streptococci: Cocci derived in one plane and remains attached to form chains of different length e.g. Streptococcus pyogenes
• Staphylococci: Cocci divide into several planes ( 3- dimensional)  resulting in the formation of irregular bunches of cells resembling a cluster of grapes e.g. Staphylococcus aureus.
• Sarcinae: Cocci divide into 3 plains at right angles to each other and resembles cubical packets.

Bacteria lacking a cell wall

1. Mycoplasm  →  Naturally occurring stable bacteria
2. L- Form →  mutant bacteria
3. Spheroplast:  cell wall damage due to toxic chemicals or antibiotics
4. Protoplast: Cell wall destroy due to lysozyme enzyme.

Note: 2,3,4 are artificially lost cell wall forms of bacteria.

Based on culture

1. Capnophilic →  CO2 loving bacteria ( 5 – 10 % CO2) e.g,  Brucella,  
2. meningococcus, gonococcus
3. Acidophilic →  Acid loving
4. Alkalophilic →  Alkali loving (pH 6.4 -9.6 ) e.g. Vibrio cholerae
5. Halophilic: salt-loving ( up to 10% NaCI) e.g. Vibrio parahaemolyticus, Vibrio vulificus
6. Microaerophillic →  Cemphylobacter jejune

A.      Bacilli or Rods

1.  Gram-positive bacilli/ Rods

Nocardia, Corynebacterium, Actinomycetes, Listeria, Lactobacillus, Bacillus anthracis, Clostridium

2.  Gram-negative bacilli / rods:

Besides those above bacilli, generally, all other bacilli are gram-negative e.g.  Hemophilus, Bordetella, Legionella, salmonella, Shigella, proteus, Escherichia, Pseudomonas, Helicobacter, Campylobacter, Klebsiella, Vibrio, Bacteroids, Pasteurella, Brucella, Yersinia, Francisella, etc.

B. Cocci

1. Gram-negative cocci: e.g. gonococcus, meningococcus, Moraxella (Branhamella)
2. Gram-positive cocci: Besides those cocci, generally all cocci are gram positive, e.g. Streptococcus, Staphylococcus, Enterococcus, Micrococcus.

C.  Classification of bacteria on the basis of flagella

• Atrichous →  No flagella e.g. Klebsiella
• Monotrichous →  Single flagella at one end
• Lophotichous →  Group of flagella at one end e.g. Helicobacter
• Amphitrichrous →  Single flagella at both ends
• Periteichous →  flagella all around the body e.g. Salmonella, E.coli

Some obligate anaerobic bacteria

• Peptostreptococcus
• Actinomyces
• Clostridium
• Bacteroides
• Fusobacterium
• Borrelia
• Treponema

Obligate aerophilic

• Neisseria
• Pseudomonas
• Aeromonas
• Leptospira

Microaerophilic

• Campylobacter

Acid-fast

• Nocardia species
• Mycobacterium tuberculosis
• Mycobacterium leprae

Note: Bacterial spores, Actinomycetes club, Cryptosporidium oocysts, are AFB positive.

Normal flora of

• Color: Bacteroids, E. coli, Clostridium, Lactobacillus, Candida albicans
•  Skin: Staphylococcus epidermidis, Candida albicans, Micrococcus, Enterococcus, etc.
•  Vagina: Candida albicans, Lactobacillus, Enterococcus.
•  Mouth: Viridans Streptococci
•  Nose: Diphtheroids, Staphylococcus, streptococcus

Capsulated bacteria @ PINK MAP

1. Streptococcus pneumoniae
2. Hemophilus influenzae
3. Klebsiella spp.
4. Neisseria meningitis
5. Bacillus anthracis
6. Clostridium perfringens

Spore forming bacteria

• Bacillus anthracis
• Clostridium

Bacterial growth

1.Lag phase

• Does not occur bacterial multiplication
• The initial phase of the bacterial growth curve
• Towards the end of the lag phase bacteria attain maximum size.
• Average duration 2 hours (1-4 hours)

2. Log phase

• Cell division occurs and their number increase
• Average duration 8 hours
• The phase of sensitivity testing
• Sporulation occurs at the end of log-phase
• the average duration is 8 hours

3. Stationary phase

• The total cell count slowly increases but the viable count remains stationary.
• Average duration few hours to few days. They are frequently gram variable and stain irregularly.

4. Decline phase

• The bacterial population starts to die although the number of total cells remains constant.
• Involution forms are frequently seen.
• Duration varies from few hours to few days.

Note

Synchronous growth: When all bacteria cells in a culture medium divide simultaneously, the growth, thus obtained is synchronous growth.

Biphasic growth: Bacteria which able to utilize two carbon sources.

Bacterial Toxins

Sterilization and Disinfection

Sterilization: Sterilization is the process by which an article or medium is freed of all living microorganisms either in vegetative form or spore state.

Sterilizing agents are:

• Heat
• Filtration
• Sterilant
• Radiation
• Sterilant  gases e.g. ethylene oxide
• Disinfection: Means destruction or removal of all pathogenic organisms.
• Disinfecting agents are:

1. Substance interfere with membrane function: e.g. surface-active agents quarternary ammonium compound e.g. Phenolic group: Phenol, cresol, and organic solvents: e.g. chloroform, alcohol
2.  Protein denaturing agents: e.g. organic acid, HCl
3. Agents that destroy or modify or modify a functional group of protein: heavy metal
4. Oxidizing agents: H₂O_2,  chlorine, iodine
5. Alkylating agents: Formaldehyde.
6. Heat: Most reliable method of sterilization
7. Dry heat: Mechanism: protein, denaturation, and oxidative damage

Types

1. Flamming: needle, cotton wool plug
2. Red heat: Inoculating wires, tips of forceps, and needle
3. Incineration: Solid dressing, bedding, pathological materials like sputum, stool, and carcasses
4. Hot air oven: Glassware, swabs, liquid paraffin, duster power, fats greases.
5. Holding period: 160 °C for 60 minutes, 170 °C for 40 minutes, and 180 °C for 20 minutes.

Sterilization control of dry heat:

• Spore of non toxigenic strains of Clostridium tetani
• Browne’s tube with green spot ( green color formation after 1 hour at 160°C)
• Bacillus subtilis subspecies niger

Moist heat:

Mechanism: Coagulation and denaturing their enzyme and structural protein.

Types

Temperature below 100°C

• Vaccine bath: 56 °C for 1 hour for three successive days
• Pasteurization of milk: Holder method → 60°C for 30 minutes and flash method → 72 °C for 15 sec
• Fractional sterilization/inspissation:  Serum or eggs containing medium: e.g. sterilization of Loeffler’s serum medium, Dorset’s egg medium, L -J medium, Ogawa medium
• Note Fractional sterilization – three consecutive days at 80 -85 °C for one hour but inspissation is 80-85 °C for one hour only

At a temperature of 100°C 

Tyndallization (3 successive days) e.g. media containing sugar or gelatin

At a temperature above 100°C

The system under pressure: Autoclave for dressing instruments, laboratory wares, media, pharmaceutical products, aprons catheters, etc.

Sterilizing temperature: 121°C for 15-20 minutes at 15 lbs pressure.

Sterilization controls: Bacillus stearothermophilus or sulfur in a test tube or Browne’s sterilizer control tube.

Filtration

For head labile liquid e.g. serum, toxin, glucose solution

Types

• Earthen- ware candles e.g.  Berkefeld and Chamberland
• Asbestos disc e.g. Seitz filter
• Sintered glass filter: used in a safety cabinet or in the cubical room.
• Membrane filter (0.45μm)
• Note: A HEPA filter is used in the safety cabinet or in the cubical room.

Radiation

1. Non – ionization: a. Infrared → syringes, b. Ultraviolet → e.g. operation theaters and laboratories
2. Ionization: a. X-ray, gamma rays ( commonly used) cosmic rays, b. Referred as to cold sterilization, c. For plastics syringes, catheter, rubber disposables, surgical catgut, bone, and tissue graft.

 Gas vapor sterilization

1. Ethylene oxide: (alkylating agents): Use: Plastic goods, Polythene tube, artery, and bone graft, cystoscope, and culture media.
2. Aldehyde: a. Formaldehyde→ wards and operation theater, heat-sensitive equipment,  woolen blankets.  b. Glutaraldehyde →Especially for tubercle bacilli c. 2% Glutaraldehyde called cidex → for cystoscope and bronchoscopes and d. Formalin – 40 % formeldehyde.
3. Hydrogen peroxide: a. Oxidizing agents-Used for contact lens
4. Halogen: a. Iodine → Tincture Iodine (Iodine + potassium Iodine + methanol),  b.Hypochlorite → 0.2 to 1 % -Uses Laboratory disinfectant on the surface of the bench and in discard spots.
5. Alcohol: 70% alcohol is used but isopropyl alcohol is better.
6. Phenol: Lysol (Cresol) 5 % disinfection of excreta, the floor of ward and operation room, sharp instruments.

Culture Media

1.  Basic Media:  Contains basic requirements for the growth of bacteria such as peptone, NaCI, water, beef extract, nutrient  e.g. peptone water → 1% peptone + 0.5 % NaCl, Nutrient broth → Peptone water + 1% meat extract and Nutrient agar → Nutrient both +2-3 % Agar
2. Enriched media: Addition of extra growth requirements such as blood or serum into basal media e.g. blood agar, chocolate agar, Dorsett’s egg medium, Loeffler’s serum medium.
3. Differential medium:  Help to differentiate bacteria into at least two groups,  e.g. MacConkey agar differentiate into lactose fermenting and non-lactose fermenting.
4. Indicator media: Contain some indicators e.g. MacConkey agar contains neutral red, triple sugar iron contains phenol red while Wilson and Blair’s media contain sulfite.
5. Selective media

• Always solid medium
• Allows growth of particular bacteria and suppressing others. e.g. Mannitol salt agar for Staphylococcus aureus
• Neisseria: Thayer martin Agar, New York city medium
• Corynebacterium: Dorset egg medium, Loeffler’s serum, Mcleod’s
• Vibrio cholerae : TCBS ( Thiosulpahte citrate bile salt sucrose) agar
• Mycobaterium → Lowenstein Jensen (LJ) medium, ogawa medium
• Beta hemolytic streptococci → Crystal violet blood agar
• H.Pylori → Skirrow campylobacter medium
• Salmonella → Willson and Blair media

6. Transport medium: Clinical specimens for culture-

• Stuart’s transport medium→ gonococci
• Cary- Blair medium → V. cholerae
• Bile peptone medium → V. cholerae
• Aims transport medium → For Neisseria gonorrhoeae  and for swab transport
• Hug much → For H. pylori

7. Enrichment media: A liquid form of selective-

• Tetrathionate broth →  for Salmonella
• Selenite F broth → Salmonella and Shigella
• Thioglycolate broth → for anaerobic bacteria
• Alkaline peptone water→ for Vibrio cholerae
• Christensen’s urea broth→ Proteus species

Important points

• The indicator used in MacConkey → Neutral red
• The indicator used in TSI → Phenol red
• The indicators used in TCBS → Thymol blue and bromothymol blue
• The indicator used in Simmon’s citrate media→ Bromothymol blue (0.2%)
• The concentration of agar in solid media → is 1.2 to 2 %
• Concentration of agar in semi-solid → 0.4 to 0.5 %
• Concentration of blood in blood agar → 5 to 10 %
• Agar is derived from red algae
• Agar coagulate at 42° C
  • L J or Ogawa contain malachite green which is inhibitory to organisms other than Mycobacterium.

Note: MacConkey agar is considered a selective differential and indicator medium.

Anaerobic culture media

1. Thioglycollate broth
2. Robertson’s cooked meat medium

Note: Mclntosh and Filde’s anaerobic jar is used.

Blood culture: For bacteremia, sepsis, infective endocarditis

• Brain heart infusion (BHI) broth
• Ratio ( 1: 10 ) blood to media
• Three samples by 1 hour of interval in infective endocarditis in 24 hours.
• Incubation at 37 °C for 7 days ( one week)

Urine culture:

• Collection: Clean catch urine (midstream urine)
• Volume: 5 ml in a sterile wide-mouthed container
•   Incubation: at 37 °C for 18 to 24 hours
•   Plating: Within 30 minutes in MacConkey  Agar (Most) and in blood agar. If the delay should be refrigerated.
•   Diameter of inoculating loop for urine → 2 mm, each loop contains 1μl of urine.
• Organism : (>10⁴ ) CFU/ ml → UTI
• (>10⁴<10⁴) CFU/ ml → correlate with clinical condition
•  < 10⁴ CFU/ ml → normal ( contaminant)
• 100 colony count equivalent to 10⁵ CFU/ml    

Sputum culture

• Blood Agar, MacConkey and chocolate Agar
• Chocolate agar is prepared by heating blood agar at 75- 80 °C for 20 minutes.
• Both X ( protoporphyrin) and V  (NAD) factors in the present in chocolate agar
• V Factor is used as an electron carrier while X factor is required for the synthesis of the respiratory enzyme.
• Chocolate agar is incubated at 37 °C at 5 to 10% CO₂ ( in a CO₂ jar) for 18 to 24 hours.
• Blood agar and MacConkey for 12- 24 hours at 37°C.
• Bacitracin (10U) disk is put in the primary inoculum of chocolate agar.
• Haemophilus is resistant to bacitracin [10 Unit].
• Optochin is placed at the secondary inoculum to differentiate Streptococcus pneumoniae from other α- hemolytic streptococci.
• Sreptococcus pneumoniae is sensitive to optochin [5 micrograms].

Staining   

Gram Stain

• Was described by Christian Gram in 1884
• Hucker’s modification is widely used

Technique

• Staining with crystal violet for 1 minute.
• Wash slide and stain with iodine for 1 minute.
• Wash and decolorize with acetone alcohol for 10-15 seconds.
• Wash and counter-stain with safranin for 1 minute.
• Wash and allow to dry it.

Interpretation

• Gram-positive → Violet or purple
• Gram-Negative → pink to red
• Yeast cell → Violet or purple ( Gram-positive)
• Epithelial cell → pale red

Ziehl- Neelsen stain/AFB stain

Technique

• Fixation(flaming)
• Staining with carbol fuchsin under the heat of 56°C for 5 minutes
• Decolorize with 3% acid alcohol for 5 minutes after washing.
• Counterstain with methylene blue or malachite green for 1 minute.

Interpretation

• Acid-fast bacilli → red ( M. tuberculosis and M. leprae)
• Background and non- AFB → blue or green ( according to counterstain used)

Reporting of AFB stain (WHO)

If ABF not seen (300 fields) report → AFB  not found

• 1 to 2 bacilli entire field → Doubtful request another sample
• 1 to 9 AFB/ 100 field → AFB seen ( with an exact number)
• 10 to 100 AFB/100 field → AFB seen (+)
• 1 to 10 AFB / field → AFB sen (++)
• More than 10 AFB / field → AFB seen (+++)

Comparison of gram stain/ AFB stain

Important point

• Alcohol fixation is preferred for suspected of Gonococci instead of flaming
• Acetone is the fastest deodorizer
• M. leprae is weakly acid-fast hence 1% v/v ( acid alcohol) or 5 % H2SO4 can be used.
• Yeast cells take a crystal violet stain.
• Some Actinomycetes, Nocardia species, bacteria endospores are acid-fast.

Special stain for-

1. Capsule: Flemming’s, Nigrosin, India ink (These are negative stains)
2. Spore: Ziehl -Nelsen method (ZN)
3. Corynebacterium diphtheriae– Albert’s stain
4. Cryptococcus: India ink
5. M. tuberculosis: Z-N (AFB)  stain, Auramine- Rhodamine staining
6. Anthrax → Sudan Black
7. Negri bodies → Seller’s stain
8. Flagella stain → Modified Leifson’s method

Antibiotic sensitivity testing

• Mueller Hinton agar (MHA)→ Commonly used
• Wilkins- Chalgren agar→ For anaerobic bacteria
• Chocolate Agar→ For fastidious bacteria like Haemophilus
• Blood Agar→ for Streptococci

Method

• Dilution method: Quantitative, by Minimal Inhibitory concentration (MIC) method or E- test.
• Disk diffusion method:

1.  Stroke’s method: Both test and control organisms are inoculated on the same plate and
2. Kirby- Bauer method: Recommended by WHO

• Test and control are placed separately
• In 9 com agar plate 6 discs are placed
• The disc is 15 mm from the edge of the plate and 25 mm from disc to disc.
• Depth of agar medium → 4 mm
• Diameter of antibiotic disc → 6 mm
• Note: Standard inoculum in matched with 0.5 McFarland standard (1% barium chloride + 1% sulphuric acid)
• 0.5 McFarland ≈150 million bacteria concentration/ml

Antimicrobial Action

Inhibitory of bacterial cell wall 

1. All penicillins (Beta-lactam agents)-e.g. Ampicillin, Amoxycillin, Piperacillin, Cloxacillin, etc.
2. Cephalosporins- e.g.  ceftriaxone, ceftazidime, cefotaxim, cefoperazene
3. Vancomycin
4. Bacitracin
5.  Isoniazide and ethambutal

Inhibition of protein synthesis

1. Aminoglycosides- e.g. Gentamycin, Tobramycin, Amikacin, Streptomycin
2. Tetracyclin
3.  Chloramphenicol
4. Macrolide- e.g. erythromycin, azithromycin, clarithromycin

Inhibition of bacterial nucleic acid

1. Rifampin
2. Actinomycin
3. Novobiocin
4. Quinolones e.g. nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin
5. Metronidazole

Inhibiting cytoplasmic membrane

e.g. colistin, polymyxin-B, amphotericin

Biochemical test of Bacterial

Catalase test

To different Staphylococcus from Streptococcus. Streptococcus is catalase-negative → 3% H₂O₂ is used for the catalase test.

Coagulase test:

To identify Staphylococcus aureus from other Staphylococcus ( Staphylococcus aureus is coagulase-positive)

Bile solubility test:

To different Streptococcus pneumonaie from other α- hemolytic streptococci (Streptococcus  pneumoniae is soluble in bile and bile salt).

Indole test:

Done by Kovac’s reagent (P- dimethyl amino benzaldehyde)

• E.coli , Klebsiela oxitoca, Proteus vulgaris, Morganella morgani etc are indole positive while Klebsiella pnemoneiae and Proteus mirabilis are indole negative.

Oxidase Test

Done by oxidase disc (1% tetramethyl p-phenylene diamine dihydro chloride)

Oxidase positive bacteria are@ PAV(BH)AN

• Pseudomonas
• Aeromonas
• Vibrio
• Brucella
• Haemophilus (except H. dureyi )
• Alkaligenes
• Neisseria

Urease test

Proteus species, Klebsiella spp., H. pylori, etc. are urease positive.

DAN ase test:

To identify S. aureus (DNA positive)

Novobiocin disc test:

To different Staphylococcus epidermis from Staphylococcus saprophyticus (S. epidermidis is sensitive to novobiocin disk)

X and V factor test:

To differentiate Haemophilus influenzae from other Haemophilus species.(Haemophilus influenzae grow around X and V  combined disc)

Biochemical Reactions of some enterobacteria and another enteric organism

Special Test for Bacteria:

1. Dick test: Streptococcus pyogenes
2. Schick Test: Corynebacterium  diptheriae
3. Satellitism test → Haemophilus infuenzae
4. Litmus mil test→ Enterococcus
5. Francis test→ Pneumococcus
6. Mac Fadyean’s Reaction→ Bacillus anthracis
7. String test→ Vibrio cholerae
8. Hanging drop preparation: Motility test(especially for V. cholerae)
9. Weil Felix Reaction: Typhus fever (caused by Rickettsia)
10. Milk ring test→ Brucella
11. Nagler’s Reaction: Clostridium perfringens
12. Wasserman’s reaction: Syphilis ( Treponema pallidum)

Motility types of different organism

1. Stately motility: Clostridium
2. Darting motility: Vibrio cholerae and Helicobacter pylori
3. spinning motility: Fusobacterium gyrans
4. Swarming motility: Proteus
5. Gliding motility: Listeria monocytogenes

General properties of medically important bacteria:

Staphylococcus

• Are gram-positive cocci in clusters in clusters mainly isolated as hospital strains.
• Staphylococcus is common in wound infections, pyoderma (impetigo), boils, abscesses, food poisoning, toxic shock syndrome.
• Mannitol salt agar (MSA) is selective medium for S. aureus.
• Toxin and enzyme produced: catalase, coagulase, staphylokinase β- lactamase, enterotoxin, toxic shock syndrome toxin, protein A.

Lab diagnosis

Note:’ Honeymoon cystitis is caused by → S. saprophyticus

Streptococci

• Gram-positive cocci in the chain, catalase negative
• Based on the group polysaccharide antigen present in the cell wall, Lancefield classifies it, 20 serogroup A to V except for I and J.

Most important species of streptococcus

• Group A β hemolytic streptococci may be further divided into 80 Griffith serotypes based on the protein antigen(MTR)
• M. protein is the major virulence factor of group A streptococci
• ASO is produced against streptolysin ‘O’ ( Immunogenic)
• Crystal violet blood agar is selective media for S.pyogens
• S.pneumonaie is Gram positive diplococci with capsule

Neisseria

Gram-Negative, aerobic, nonmotile, oxidase-positive diplococci, intracellular or extracellular, common pathogenic group.

1. N. meningitidis( meaningococci)

Half-moon shaped capsulated cause- meningitis

2. N. gonorrhoeae (gonococci)

Bean-shaped non-capsulated cause gonorrhea ( Whitish discharge) from the urogenital tract.

Selective media

Thayer martin medium with VCN.

Modified, New York city medium

Corynebacterium diphtherriae

• Also known as Klebs – Loeffler bacillus
• Gram-positive, non-motile, club-shaped, non-sporing, non-capsulated bacilli
• Appears as a Chinese letter
• Disease: Nasopharyngeal tonsilar Diptheria. ( Formation of the grey yellow pseudomembrane around the tonsil)

Selective media:

Tellurite Blood Agar

Loeffler’s Serum medium

Satin: Albert’s stain (metachromatic granules)

Toxigenicity Test: Elek’s Gel Precipitation Test, Schick test.

Clostridium

• Obligate anaerobic gram-positive spore-forming bacilli.
• Motile except CI. perfringens and CI. tetani type IV species.
• CI tetani → tetanus ( trismus or luck jaw disease)
• CI. perfringes= CI. Welchi → Food poisoning, gas gangrene.
• CI.botulinum→ Botulism
• CI. → Pseudomembranous colitis, (Antibiotic-associated diarrhea)

1. ” Tennis racket” morphology is the feature of CI. tetani.

2. Antibiotic-associated diarrhea → CI. difficle

3. Nagler’s Reaction → By CI. Perfringens

Media: 

Robertson’s cooked medium (best)

Thioglycolate medium

Vibrio cholerae

Gram-negative, oxidase-positive, short curved, cylindrical rod, comma-shaped, motile with single polar flagellum, showing Darting motility.

Route of infection (ROI)- faeco oral Route.

The most prominent pathogen to man is

1. V. cholerae → causes cholera
2. V. parahaemolyricus → Halophilic ( 7 %) – food poisoning
3. V. vulnificus → lactose fermenting Vibrio cause wound infection.

(V. Parahaemolyticus show Kanagawa phenomenon.)

Laboratory diagnosis

• Collection of stool specimen with the help of ”cholera catch device ” or screw-capped container.

Transport media

• Venkataraman Ram Krishnan (VR) media
• Alkaline peptone water (pH 8.5) for 4-6 hours
• Cary and Blair Medium

Selective media

TCBS (Thiosulphate- citrate bile salt sucrose agar) Large yellow ( source fermenting), Colony which may become green on continued incubation. ( late lactose  fermenting)

Biochemical character:

• Late lactose fermenting
• String test (0.5% sodium dexoicholate) → positive
• Cholera red reaction – positive

Note: ” Rice water” stool is characteristic of cholera.

Pseudomonas

• Aerobic, Gram-Negative, Motile ( except Pseudomonas mallei) by means of one more polar flagella.
• Non-spore-forming, strictly aerobic, oxidase-positive( except P. maltophilia)
• Fruity smell and the pigmented colony is the characteristic of Pseudomonas.

Selective Media

Cetrimide agar

Pigment: Pyocyanin, pyorubrin, pyoverdin, Pylomelanin

 Mycobacteria

• Gram-positive, acid-fast non-motile, non-capsulated, and non-sporing fungus-like bacteria
• GC content of DNA → 62-72 mol% (M. leprae-55 mol%)

Pathogenic group

1. M. tuberculosis
2. M. lepae
3. M. bovis

• Jones’s bacillus→ M. paratuberculosis
• Hansen’s disease→ Leprosy( M. lepare)
• Rapid Grower → M. Chelonei and M. fortuitum

Staining

Z.N stain, rhodamine- auramine stain.

• The concentration of sputum Smear → Petroff’s method

Culture

• L-J medium
• Ogawa medium
• Dorset egg medium

Antibiotic sensitivity/ biochemical test and preparation of antigen and vaccine from

• Davis and Dubos medium
• Middle Brooke medium.

Cocktail of antibiotics

• To prevent overgrowth by contaminants @ PANTA
• P- Polymyxin
• A- Amphotericin B
• N- Nalidixic acid
• T- Trimethorpin
• A- Azocillin
• Incubation time for culture → 6 weeks
• BACTEC  system → in one week
• Colony: Rough, buff, and tough
• Confirmatory test → Niacin positive

Mantoux test ( Tuberculin skin test)

• Intradermal injection of 0.1 nL (nanoliter) PPD ( purified  protein Derivative) into the flexor  aspect of the forearm
• Read the result after 48-72 hours of injection.

Induration with erythema

> 10 mm → positive

6 to 10 mm → equivocal

< 5 mm → negative

Sputum microscopy-3 samples

1st sample 

spot

80 %

2nd sample

morning

(+15%)

3 rd sample

spot

(+5% → sensitivity)

Note: New guidelines suggests only two specimens.

For positive sample

Sputum smear minimum contain 10000 bacilli/ ml

Sputum culture → 10-100 bacilli / ml

PCR → 1-10 bacilli/ ml

Therapy (HRZE)

Bactericidal

1. Isoniazid (H)
2. Rifampicin (R)
3. Pyrazinamide (Z)
4. Streptomycin

Bacteriostatic-Ethambutol (E)

• A combination of drugs- HRZE is given for 6 months.
• In MDR (multi drugs Resistant)→ Isoniazid and Rifampicin are Resistant.

Spirochetes

Medically important spirochetes are

1. Treponema
2. Leptospira
3. Borrelia

• T. pallidum is the causative agent of syphilis.
• Spiral due to endo flagella

1 . Reiter’s -strain → Non-pathogenic

2. Nichol’s strain → pathogenic

Antigen- 2 types

1. Group-specific Antigen → All pathogenic and non-pathogenic
2. Non-specific antigen

Reagin Antibody

Appears in the blood of syphilitic patients that reacts with lipids hapten of the bovine heart (Ag). This lipid hapten Ag is chemically phosphatidylglycerol and known as cardiolipin.

The reagin Ab can be produced by other conditions like

1. Acute febrile illness
2. Pregnancy
3. Connective tissues diseases
4. Leprosy
5. Malaria

Test for reagin Ab

• Wasserman rection
• Kahn test
• VDRL (venereal Disease Research Laboratory)
• RPR (Rapid Plasma Regain)

Syphilis

• Disease due to sexual contact or congenital
• Incubation period 10- 90 days

Stage

• Primary stages  → hard chancre
• Secondary stage  → maculopapular rash
• Tertiary stage → gumma ( granulomatous) nodules in skin and mucus membrane

Lab diagnosis

•  Darkfield microscope ( > 10 power 4 spirochetes/ ml)
• Fontana’s stain ( silver impregnation)
• Warthin – Starry stain

Specific Test for Treponeme

• FTA – ABS ( Fluorescence Treponemal antibody absorption test)  → (most specific).
• TPHA ( Treponema pallidium haemagglutinaion)
• TPI ( Treponema pallidum immobilization)

” yaws” disease  → by T. pertenue

“Lyme”  disease  → Borellia burgdorferi

”Pinta” Disease  → by T. caretenum

Different between VDRL and RPR

Brucella

• Cocobacilli, aerobic, gram negatives, non-motile, non-sporing, non-capsulated.
• Brucellosis is essentially an infection of animal ( zoonotic disease)

Main species

B. melitensis  → sheep, goat  → ( malta fever)

B. abortas  → cattle: → undulant fever.

B. suis  → Pig

Mode of infection (MOI)

1. Consumption of infected milk and milk production.
2. Direct contact with farmworkers, veterinary surgeons who come in frequent contact with infected animals.

Culture

Castaneda’s method of flood culture.

Trypticase soy broth

”Milk ring” test is done for detection of animal infection.

Haemophilus influenzae

• Gram-negative, pleomorphic, or coccobacillus, occasionally filamentous form, oxidase-positive.

Pathogenesis

Otitis media, conjunctivitis, lower respiratory tract infection, meningitis, epiglottitis.

• Mainly pathogenic is H. influenzae type-b
• In Nepal 40% LRTI and 60 % meniagitis are due to H. influenzae.

Culture media

Levinthal broth/agar

Chocolate agar

Columbia agar

Identification

Satellitism test → positive (Best in Pigeon Blood)

Grow on X and V factor ( combined) rich region in MHA.

Note:

X -factor is required for the synthesis of the respiratory enzyme, which can be obtained from protoporphyrin IX/ Haem.

Properties of different Haemophilus spp.

Note: Soft chancre is caused by by- H. ducreyi

Bordetella

Gram-negative, coccobacillus, non-mobile, non-sporing, capsulated, strick aerobes.

B. pertussis  → Causative agent of whooping cough ( in children)

Culture media

Bordet – Gengou’s medium

Cephalexin charcoal Blood Agar

Colony: Mercury or dewdrop appearance

Vaccine: DPT ( Diptheria  Pertussis and Tetanus)

Actinomycetes

• Ray like: Ray fungus
• Gram(+)ve, non motile/ non acid-fast
• Grow preferably under anaerobic conditions in the presence of CO₂ 
• ”Sulphur granules”  are found in Actinomycetes granulomatous infection.
• Colony: Spidery colonies ( i.e. small creamy, grey-white  rough nodular surface.)

‘Club’s are acid-fast

Culture: Thioglycollate broth

Rickettsiae

• Gram-negative, short rod, and pleomorphic
• Obligate intracellular

Staining

Macchiavello’s stain

Culture: Hella cell, Yolk sac of chick embryo

Serological Test: Weil Felix reaction

Chlamydiae

• Nonmotile, gram-negative, obligate intracellular
• Stain: Machiavelli’s stain and Gimenez stain
• Also called energy parasite
• Two forms

1. Reticulate bodies: larger

2. Elementry bodies: smaller

Culture

Hella cell lines, monkey kidney cell lines, yolk sac inoculation.

Mycoplasma

• The cell wall deficient organism
• Also called pleuro pneumonia-like organism (PPLO)
• Organisms are seen like fungus in the form of branching filaments.

Culture

PPLO Agar

PPLO biphasic broth/ agar

These media are supplied with 20 % horse serum, yeast extract, and DNA

Colony: fried egg appearance

Stain: Diene’s stain( royal intense blue)

Disease: M. pneumoniae → Atypical pneumonia/walking pneumonia

Bacillus

• Motile with peritrichous flagella except for anthrax bacilli. (nonmotile)
• Are spore-forming gram-positive bacilli and capsulated
• Most common contaminants of bacteriological culture media.
• The most important species are:

1. B. antracis
2. B. cereus

B. anthracis

• Selective medium: PLET medium
• On agar plate: frosted glass appearance
• Solid media with Penicillin: a string of pearl reaction
• In transmitted light: cut glass appearance
• Bacilli arranged in Bamboo stick/box car-like appearance
• Shows Mac Fadyeans reaction on staining with polychrome methylene blue.
• Spore is used as bioterrorism.

Enterobacteriaceae

A heterogeneous group of a large number of important gram-negative bacilli, which are strictly found in the intestine.

• Gram-negative
• Non-spore forming
• Aerobic or anaerobic
• Motile except Shigella and Klebsiella
• Capable of fermenting glucose or another carbohydrate with or without gas production
• Oxidase negative
• Catalase negative ( except Shigella dysentriae type-1)
• DNA base ratio: 50-59 moles%

Classification

The tribe I- Escherichia

Genus:

• Escherichia
• Edwardsiella
• Citrobacter
• Salmonella
• Shigella

Tribe II- Klebsiella 

Genus:

• Klebsiella
• Enterobacter
• Hafnia
• Serratia

Tribe-III- Proteae

Genus:

• Proteus
• Morganella
• Providencia

Tribe IV – Erwinaie

e.g. Genus: Erwinia

Tribe V- Yersiniae

e.g. Genus: Yersinia

Escherichia coli

• Gram-negative, motile except (E- blate) by Peritrichus flagella, non-spore-forming, lactose fermenting, and Indole(+)
• IMVC →++–
• The most causative agent of UTI (>70%)

Also, cause diarrhea such as

• Enterotoxigenic E. coli ( ETEC)
• Traveler’s diarrhea
• Produce enterotoxin: LT and ST
• Biken’s test, or Rabbit ileal loop test in done for LT

Enteropathogenic E.coli (EHEC)

• infantile diarrhea

Enterohaemorrhagic E. coli (EHEC)

• life-threatening hemorrhagic diarrhea
• produce Shiga  like a toxin

Entero Invasive E.coli (EIEC)

• Caused dysentery similar to shigellosis
• Diagnosis ‘Sereny test’

Salmonella

• Gram-negative, non-sporing, non-capsulated, motile except Salmonella gallinarum and pullorum (non-motile)
• MOT → fec0- oral route
• Infective  done: 10 bacilli through ingestion

Diseases

A. Enteric Fever/ Typhoid fever

• S. Typhi
• S. Paratyphi A, B, and C

B. Food poisoning/gastroenteritis

e.g. S. Typhimurium

Classification: Kauffman and white

Lab diagnosis

BASU

B → Blood culture → 1 st week

A→ Antibody detection → 2nd week

S→ Stool culture → 3rd week

U→ Urine culture → 4th week

Blood culture

• Castaneda’s method prefer
• BACTEC system → early detection

Media/Broth

BHI: Brain Heart Infusion

The ratio of blood to Broth: (1:10)

i.e 5ml blood+ 45ml broth

Incubation: at 37°C for week

Agglutination test

• Widal test
• Done in Kahn tube

H- agglutination: wooly clumps

O- agglutination: disk-like granules deposit in the bottom of the tube

‘H’ Ag is prepared by adding 0.1% formation to a 24 hours broth (Hajana broth)

‘O’ Ag is prepared by culturing organism on Phenol Agar( 1:800)

Stool culture

For carrier detection

Media

A. DCA( deoxycholate Citrate Agar)

B. Wilson and Blair media (Selective)

Enriched media

Tetrathionate broth

Selenite F broth

Shigella

• The causative agent of bacillary dysentery
• Infection dose: 1-100 bacilli

Culture

S-S Agar

XLD Agar

DCA Agar

• Colicin typing in done
• Sereny test for the toxin.

Contd…