MICROBIOLOGY

Structure and Function:

 Prokaryotes (bacteria) lack nucleus, nuclear membrane and organelles like
mitochondria and Golgi bodies.
 Prokaryotes have cell wall (which is unique to them as eukaryotes lack cell wall)
 Gram-negative cell wall has Lipopolysaccaride (LPS), which functions as endotoxin.
Therefore only gram-negative organisms can form endotoxin.
 Gram-positive cell wall has techoic acids.
 Both gram positive and gram-negative organisms can produce exotoxins
 Maximum exotoxin production is seen in log (exponential) phase of bacterial growth
curve.

Flagella are organs of locomotion (motility) in bacteria:

 Monotrichous- Vibrio cholera- darting motility
 Amphitrichous- Listeria- tumbling motility
 Lophotrichous- Helicobacter
 Peritrichous- Motile members of Enterobacteriaceae family e.g. E. coli, Salmonella,
Proteus, which show stately motility
 Endoflagellum: Spirochaetes: corkscrew motility
 Twitching motility in Eikenella is because of pili
o Pili (fimbriae) are means of adhesion
o Bacterial genetics
o Specialized pili (sex pili) carry out conjugation (transfer of genetic material from
one bacterium to another.

A. Transformation: acquisition of new genetic material through naked DNA

demonstrated in pneumococci by Griffith experiment
B. Transduction: transfer of genetic material into bacteria by bacteriophage (virus)
C. Transposon- jumping genes- discovered by Barbara McClintok- Nobel prize

Staining methods:
 Gram staining: differential staining
 Ziehl Neelson staining: acid fast staining- Mycobacteria, nocardia etc
 Kinyoun staining: cold acid fast staining: Cryptosporidium parvum
 Albert staining: metachromatic staining- Corynebacterium diphtheriae
 Giemsa, Leishman, Wright, Field, JSB stains: Romanowsky stains

Growth Requirements:
 Obligate Aerobes: grow only in presence of oxygen eg Bacillus
 Facultative Anaerobes: grow better in presence but can grow in absence of oxygen as
well eg. Enterobacteriaceae family
 Obligate Anaerobes: grow only in absence of oxygen eg. Clostridium
 Microaerophilic: grow best in 3-5% oxygen eg Helicobacter
 Capnophilic: grow well with additional 5-10% carbon dioxide eg. pneumococci,
meningococci, H. influenzae

Culture media:
 Transport media: Cary Blair medium (universal transport medium)
o VR fluid- Vibrio cholera
o Stuart’s transport medium: respiratory specimen
 Enrichment media: Selenite F broth- Salmonella, Shigella
o VR fluid, Alkaline peptone water- Vibrio cholera
 Enriched media: Blood Agar, Chocolate agar
 Selective media: Wilson Blair medium: Salmonella
o Potassium Tellurite Blood Agar: Corynebacterium diphtheriae
 Medium for Mycobacteria: Lowenstein Jensen medium, Middlebrook medium
 Medium for Corynebacterium: Loeffler’s serum slope

 Antibiotic susceptibility testing done by diffusion methods or dilution methods

 Disc diffusion methods: Stokes method, Kirby Bauer method
 Dilution methods: for calculating minimum inhibitory concentration

Serological Tests:
 Slide agglutination- blood grouping, serotyping of Salmonella
 Tube agglutination- Widal test
 Slide Flocculation- VDRL
 Tube Flocculation- Kahn test
 Complement fixation test- Wasserman test

Molecular tests:
 Polymerase chain reaction: amplification method, done in thermocycler.
o Steps- denaturation (94-96°C), annealing (54°C) and extension (72°C).
Enzyme used Taq polymerase. Gel stained by ethidium bromide.
 Multiplex PCR: diagnosing many organisms in one PCR e.g. CSF sample
 Nested PCR: increases specificity
 Reverse Transcriptase (RT)-PCR: quantitation
 Arbitrarily primed PCR (AP-PCR) or Randomly Amplified Polymorphic DNA
(RAPD)- used for epidemiological typing or strain differentiation
  Ligase chain reaction (LCR): amplification method, does not require
thermocycler
 b-DNA: quantitation method without thermocycler
 Pulsed Field gel electrophoresis (PFGE): best method (gold standard) of
epidemiological typing

Sterilization and Disinfection:

 Hot air oven: 160°C for 1 hour. Glassware, oils, wax, powders, sharp instruments
 Autoclaving: 121°C for 15-20 minutes at 15 pounds per square inch pressure.
Gloves, masks, gowns, linen, cotton, rubber plastic containing articles, surgical
instruments, catheters, agar
 Inspissation: not a method of sterilization. Only a method of solidifying media
containing serum (LSS) or egg (LJ) (80°C for 30 minutes)
 Tyndallization: method of sterilization for media containing egg or serum. (80°C
for 30 minutes for three consecutive days with intermittent overnight incubation
at 37°C)
 Filteration: for heat sensitive fluids. Vaccines, antibiotic solutions, serum, sugar
containing media
 Low temperature Steam formaldehyde (LTSF), Gas Plasma: newer methods for
heat sensitive articles
 Bulk sterilization- Gamma radiation or ethylene oxide
 UV rays- not able to sterilize, just surface disinfectants
 Chemical sterilization- Glutaraldehyde (2% Cidex) for endoscopes
 Universal disinfectant: Hypochlorite (bleaching agent)
 Biological indicators of sterilization- Bacterial spores (Bacillus
steareothermophilus, Clostridium tetani var. niger)

A. STAPH. AUREUS:
 M/cc of Pyoderma
 M/cc of breast abscess
 M/cc of acute osteomyelitis (hematogenous spread)
 M/cc of Pyomyositis.
 M/cc of nosocomial pneumonia Staphylococcal scalded skin syndrome.
 Food poisoning(milk & milk products),Toxic shock syndrome
 Folliculitis , Furuncle, Carbuncle , Stye
B. STAPH. EPIDERMIDIS:
 M/cc of catheter related blood stream infection.
 M/cc of prosthetic wall endocarditis.
C. STAPH. SAPROPHYTICUS:
 M/cc of UTI in female of child bearing age group {over all mcc of UTI E.coli}
D. STREP.VIRIDANS:
 M/cc of dental caries
 M/cc of SABE
E. STREP. PYOGENS: (Group-A beta haemolytic streptococci)
 M/cc of impetigo M/cc of cellulitis
 M/cc of necrotizing fasciitis.
 Scarlet fever (sore throat + skin rash)
 Erysipelas (sharp rash +no sore throat)
 M/c bacterial cause of sore throat.
 Acute rheumatic fever Acute PSGN
F. STREP.AGALACTIAE: (Group B beta-hemolytic streptococci)
 Neonatal septicaemia
 Neonatal meningitis
G. STREP.PNEUMONIAE:
 M/cc of community acquired pneumonia
 M/cc of lobar pneumonia
 M/cc of acute meningitis in adults
 M/cc of acute otitis media in children
 M/cc of severe infection in splenectomised patients

Types of Motility & Organism:

 Shooting star /darting Motility - Vibrio cholera
 Tumbling Motility – Listeria
 Gliding Motility – Mycoplasma
 Cork Screw Motility - Treponema Pallidum
 Swarming Motility - Clostridium Tetani, Proteus
 Leaf like Motility - Giardia lambia
 Twitching / jerking Motility – Trichomoniasis

Virus Family:
 Measles – RNA Paramyxovirus
 Rubella – RNA Togavirus
 Mumps – RNA Myxovirus
 Influenza – Orthomyxovirus
 Crimean congo fever – Nairavirus - bunya virus
 Rabies – Lyssavirus genus of Rhabdovirus
 Dengue – Arbovirus
 Ebola Virus – Filoviridae

Culture Media:
  Staph.Aureus - Mannitol salt agar; Ludlam’s medium
 Streptococcus - Crystal Violet blood agar
 Cornyebacterium - Loeffler’s serum medium; Potassium tellurite agar
 Pseudomonas - Cetrimide agar
 Mycobacterium - Lowenstein Jensen (L-J medium) or Dorset egg medium
 Bordetella - Bordet Gengue medium
 H.Pylori - Skirrow’s medium

Hot air oven:

 Temperature 160°C for 2hrs
 Materials sterilized: Glass, forceps, liquid paraffin, fat, dust powder

Inspissation:
 At 80 C for 30 min for consecutive 3 days.
 Useful for Egg based media.

Autoclave:
 121 C for 15 min at 15 psi pressure.
 Sterilization control – Bacillus stearothermophillus.

Sterilization:
 Prions by-
 Autoclave at 134°C for 1 -1.5 hr
 0.5% hypochlorite for 2hrs
 NaOH for 1 hr
 Metallic surgical instruments – Autoclave or Hot air oven.
 Operation theaters, wards, lab – formaldehyde gas > UV Rays
 Cystoscope, bronchoscope – Orthophthaldehyde > 2% glutaraldehyde (Cidex
solution)
 Sharp instruments- Cresol
 Plastic syringe, catheters, tissue grafts, dressing – ionizing radiation (Cold
Sterilization)
 Respiratory machines – Ethyline oxide

 Water : Chlorine as 0.2% hypochlorite

 Weil-felix test: Rickketssia
 Microscopic agglutination test: Leptospira
 Paul bunnel test :EBV
 WIDAL TEST : Enteric fever
 Standard Agglutination test: Brucella
  Schick test : diphtheria toxin
 Wasserman test : syphilis

1. Echinococcus Granulosus:
a. Definitive host: Dog
b. Intermediate host: Man
c. Infective stage: Egg
d. Test: Casoni’s test (Type 1 HS)
2. Diphyllobothrium latum( Fish tapeworm)Largest tapeworm.
a. Definitive host: Man
b. Intermediate host: First- Cyclops, Second- fresh water fish
c. Infective stage: Plerocercoid (Stage III Larva) causes Megaloblastic anemia
(Absorbs Vit. B12)
d. Single hostInfectious stage: Egg
3. Hymenolepis nana (Dwarf tapeworm)
4. Schistosoma hematobium (Blood Fluke) – CANCER ASSOCIATED resides in
visceral & pelvic plexus A/w: Bladder Carcinoma, Hematuria, Hydronephrosis
5. Schistosoma mansoni resides in mesenteric veins, Draining sigmoido-rectal region
a. C/P: Swimmer’s itch (Type I HS)
b. Katayama fever (Type III HS)
c. Technique: KATO KATZ Thick smear technique
6. Fasciola hepatica (Sheep liver fluke)Definitive host: Sheep or man
a. C/M: Hepatomegaly, Halzoinm bile duct obstruction
7. Paragonimus westermani (Lung fluke)
a. Definitive host: Man
b. Intermediate host: 1 st snail, 2nd crey/crab fish
c. Mode of transmission: Ingestion of crab fish
d. C/M: Cyst in Right lung
o Golden brown sputum
o Endemic haemoptysis.
o Operculated eggs in coughed sputum
8. Clonorchis sinunsus (Chineese liver fluke) – CANCER ASSOCIATED
a. Definitive host: Man
b. Intermediate host: 1 st snail, 2nd crey/crab fish
c. Mode of transmission; Ingestion of crab fish
d. C/M:Cholangitis , Cholangio carcinoma
9. Nuerocycticercosis:
o Most common parasitic CNS infection of man.
o Most common cause of adult onset Epilepsy.
o Most common site: Subarachnoid Space.
 o IOC: CT/MRI
o Criteria: Del Brutto’s criteria
o Treatment: Albendazole, Praziquantel.
10. Parasites causing anemia:
o Hookworm: Iron deficiency anaemia.
o Trichuris trichura: Iron deficiency anaemia.
o Diphyllobothrium latum: Megaloblastic anaemia.
11. Parasites causing Malignancy:
o Opisthorchis viverrini: Cholangia carcinoma of bile duct
o Chonorchis sinunsus: Cholangio carcinoma of liver, bile duct & Adenocarcinoma
of pancreas.
o Schistosmia hematurium: SCC of Urinary bladder
12. Non-bile stained eggs (NEHA):
o Necator americanus
o Enterobius vermicularis
o Hymenolepsis nana
o Ancylostoma
13. Man is intermediate host of ( STEP):
 Sacrolytis lindemanii
 Toxoplasma gondii
 Tenia solium
 Echinococcus granulosus
 Plasmodium
HACEK group of organisms
o H-haemophillus species
o A-actinobacillus actinomyecetemcomitans
o C-cardiobacterium hominis
o E-ekinella corrodens
o K-kingella knigae