Understanding Microbial Staining and Sterilization Methods

Chanting Lines

Gram Staining

• Primary stain is crystal violet. 
• Iodine (Mordant) is a fixer. 
• Safranin is a secondary stain. 
• Gram+ve organisms are purple in color. 
• Gram-ve organisms are red in color. 

ZN Staining

• Carbol fuchsin is red in color. 
• Acid / Acid alcohol 
• Methylene blue/malachite green—background color. 

Concentration of Acid Alcohol 

• H₂SO₄ – 20% 
• Lepra: – Acid fast (5% H₂ SO₄) known as Fite faraco stain. 
• Nocardia, legionella: 1% H₂SO₄ 
• Spores, head of sperm : 0.25-0.5% H₂SO₄

Stain for Spores is Shaffer and Fulton Stain. 

• Malachite green-spare. 
• Heat Dormant 
• Water decolorizes. 
• Safranin Red – Organism.

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Spores

Types of Spores 

• Based Upon bulge 
• Non bulging – bacillus. 
• Bulging – Clostridium

Based Upon Location

• Subterminal – It is the most common location. 
• Terminal: Clostridium tetani (looks like a drum stick) & Clostridium tetani (looks like a tennis racket). 
• Central – Bacillus cereus & Clostridium bifermentans.

Stains 

Albert stain: 

• Used for volutin/babes Ernst granules present in C. diphtheriae.
• Located as 2 poles and also called as bipolar granules. 
• Metachromatic Granules-stained by toluidine blue. 
• It contains polymetaphosphate; it is an energy source. 

AlberSolutions 

1. Toluidine blue (metachromatic stain, 2 colors).
2. Malachite Green: organism. 
3. Iodine

Stains for Volutin Volutin Granules are:

• Ponder's stain 
• Loeffler methylene blue (best) 
• Albert stain 
• Neisser stain

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Maximum Magnification Offered By A Light Microscope Is 1000x.

• The condenser is placed below the stage.

Labeling of Light Microscope

• Light 
• Condenser 
• Coarse adjustment knob 
• Fine Adjustment knob 
• Two lenses.
  • Eyepiece lens
    • Magnifies 10 times. 
  • Objective Lens
    • Scanner Lens – Magnifies for 4 times. 
    • Low Power - Magnifies 10 times. 
    • High Power - Magnifies 40 times. 
    • Oil Immersion – Magnifies 100 times.

Light Type (Light used in these microscopes) 

• Light microscope: light used is transmitted light. 
• Dark field microscope: reflected light 
• Fluorescent microscope: the light used is ultraviolet light. 

Fluorescent Microscope 

• Light is ultraviolet light. 
• Dichroic Missos are used.
• Different Dyes.
  • For TB, Dye used is auramine and rhodamine. 
  • For fungus, the dye used is calcofluor white; it is a fluorescent stain that binds to calcofluor white or the cell wall. 
  • For Malaria, the dye used is Acridine orange. Adding Acridine orange to malaria is a technique, and this technique is known as 0. 
  • For flow cytometry, we also use a dye called fluoro isothiocyanate. 

Dark field Microscope 

• Used for spirochetes (spiral structures). 
• Medium is vacuum. 
• No light is used. 
• An electron beam is used – the electron is enclosed. 
• A metallic slide is used. 
• 2-2.5% Glutaraldehyde is used as a fixative. 
• Embedding media is resin.

Bacterial Growth Curve: 4 Phases

• Lag phase 
• Log phase
• Stationary phase 
• Dead phase 
• Maximum size of the bacteria seen in the Lag phase. 
• Cell division is going to occur in the log phase. 
• Spare formation occurs in the stationary phase. 
• Also the toxin production, Antibiotic production & Bacteriocin production happen in the Stationary phase.

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Sterilization Methods Gas sterilization 

• Control (Bacillus Globigii)
• Gas used ethylene oxide ETO
• Used for Cardiopulmonary machines

Radiation 

• Control (Bacillus Pumilus)

Filtration

• Average size of a filter is 0.22 m 
• Control (Brevundimonas diminula and Serratia).

Plasma Sterilization & Autoclave

• The chemical used is H₂O₂, it is a low temperature technique. 
• Arthroscopes and ureteroscopy done by plasma sterilization 
• Control is Bacillus stearothermophilus 
• Sterrad is a machine used for plasma sterilization.

Hot airOven

• Uses – Glass wall. 
• Control – Bacillus subtilis, Clostridium tetani, & Bacillus atrophaeus C and spore-forming organisms can be used.

Toxins

Toxins – decreasing protein synthesis are Diphtheria 

Toxin and exotoxin of pseudomonas and Shiga toxin and verocytotoxin (Shiga-like toxin) 

• Diphtheria toxin and exotoxin cause ADPRibosylation of EF2 & decreased protein synthesis. 
• Shiga toxins and verocytotoxin act on 60s ribosomes & decrease protein synthesis. 

Toxins that Act by Increasing cAMPand cGM`

Toxins increasing cAMP

• Cholerae (rice water stools). 
• Anthrax 
• Enterotoxigenic E.coli 
• Pertussis 

Enterotoxins have two toxins

• Labile toxin (Going to act by cAMP) 
• Stable toxin (Going to act by cGMP) 

Organism work on both is Bacillus cereus. 

• Emetic is cGMP 
• Diarrheal is cAMP

Also read: Antimicrobial Drugs: Combatting Infections

Toxic Shock Syndrome

• The organism is Staphylococcus aureus. 
• Toxin is enterotoxin F or TSST 
• It is a fatal disease and associated with long-term tampon usage.

Tetanus

Have two Tetanus exotoxins.

• Tetanolysin (O₂ labile) 
• Tetanospasmin (O₂ Stable)

Types of Motilities

1. Tumbling + Differential motility (listeria). 

• Motile at 22-25º C 
• Non-Motile at 37ºC 

2. Darting/shooting star (Vibrio and campylobacter jejuni) grow at 42ºC 
3. Swarming (proteus, Vibrio parahaemolyticus, clostridium tetani, Serratia). 
4. Falling leaf (Giardia lamblia) 
5. Twitching (trichomonas vaginalis) 
6. Corkscrew motility (Treponema) (Tight coils) 
7. Lashing (Borrelia) (Loose Coil)

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Culture Media

• Selective media for Staphylococcus is mannitol salt agar. 
• The color is yellow. 
• The transport media is pike's medium. 
• Blood agar finding – wide zone hemolysis. 
• Selective media also include crystal violet blood agar & PNF
• Streptococcus Agalactiae 

Streptococcus Pneumoniae

• Shows Carrom Coin Draughtsman Colonies.

Enterococcus

Significance of resistance to 40% bile

• Culture media – Bile aesculin Azide Agar. 
• Enterococci causes aesculin lysis. 
• Results in Black color. 

Neisseria

• Transport media –Stuart's medium and Amies medium. 
• Growth – Thayer Martin Medium.

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Corynebacterium Diphtheriae

Loffer's serum slope is used. 

• Kleb Loffer's media 
• Enriched media 
• Grows in 8 hours. 
• In case of selective media 
• Potassium tellurite agar is used. It takes 48 hours.

Bacillus Anthrax

• Appearance in nutrient agar is classical medusa head appearance. 
• Gelatin agar—the appearance inverted for tree appearance. 
• Selective media (PLET)

Bacillus cereus: Chinese fried rice. 

• Culture media (MYPA, PEMBA)

Clostridium Tetani Perfringens 

• They are anaerobic organisms. 
• Culture media is Robertson-cooked meat broth. 
• Clostridium Difficile – CCYA, CCFA are the culture media 
• Vibrio – Transport media for vibrio is Cary Blairmedium. And VR medium. 
• (TCBS) sucrose fermentation is seen by Vibrio cholerae.

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Fried Egg Colonies are seen in Myoplasma/Eaton's Agent 

• Seen on PPLO Agar. 
• The stain used is DIENE'S Stain. Coded on the surface of the culture. 

Dimorphic Fungi Blastomycosis 

• Blastomycosis : Also known as North American blastomycosis. Gilchrist or Chicago disease.
  • Organism – Blastomyces Dermatitidis. 
  • Size – 8-15 microns. 
  • Classical figure of 8 appearance.
• Histoplasmosis – Also known as Cave's disease/Ohio disease, Mississippi disease & darling disease
• Appearance – Tuberculate macroconidia.
• Penicillium marneffei – Also known as Talarmyces Marneffei.

• The natural reservoir is Bamboo rats. 
• Appearance – Brush/Broom conidia morphology is seen. 
• A red pigment is formed. 
• Paracoccidioidomycosis

• Also known as South American blastomycosis. 
• Appearance - Mickey mouse appearance & Pilot/Marines wheel appearance. 
• Coccidioidomycosis 

• Also known as dessert rheumatism, valley fever, or California disease. 
• It contains barrel-shaped arthrospores.
• Sporotrichosis 
  • Also known as Gardner's disease/lymphocutaneous disease.
  • Rosettes of conidia appear. 
  • Star-shaped bodies (asteroid bodies) are seen in sporothrix schenckii. (extracellular) and sarcoidosis (intra cellular).

Also read: Fungal Infections: Types, Causes, Symptoms, Diagnosis & Treatment

Viruses:

• Smallest virus – Parvo virus (20 m)
  • Only DNAvirus which is a single standard. 
• Largest virus – Pox virus (300 nm)
• RNA viruses: have one copy of SS RNA, which is unsegmented except. 

• Retrovirus – has 2 copies. 
• Reo/rotavirus- has ds RNA

• Aplastic crisis in haemolytic anemia→ Parvovirus B19.
• Hemolytic crisis is a WEBV.

Downey Cells: 

• Origin is CD8T lymphocytes.

A test for EBV (Epstein-Barr virus) is a monospot test. 

• Decoy cells are seen in post transplant patients 
• Caused by bad kidney virus (BK virus) 
• Koilocytic atypia is noticed with human papilloma virus 
• E₆ & E₇ are responsible for carcinogenesis as E₆ inactivates the p53 and E₇ inactivates the RB gene, causing cancer. 
• L₁ capsid protein is used for making a vaccine. 

Agglutination Tests 

• The organism for the cold agglutination test is mycoplasma. 
• Organism for standard agglutination test is brucella 
• The organism for the microscopic agglutination test is Leptospira. 

Also read: Helminthology : Structure, Classification, Growth, and

Dimension

• If a reaction occurs in the test tube, one dimension. 
• Its reaction occurs in slide-two dimensions. 

Diffusion 

• Single diffusion: only antigen moves. 
• Double diffusion: both antigen and antibody move.

One Dimension 

Mnemonic: OOTD 

O - Single diffusion (Oudin test) 

O - Double diffusion (Oakley-fulthorpe test) 

T- Tube 

D - Dimension (ID) 

• Double diffusion in 2D – Ouchterlony test E.g. Elek's Gel precipitation test. 
• Single diffusion in 2D—radial immunodiffusion.

Marrack lattice Hypothesis

• Zone of equivalence 
• Antibody is equal to antigen 
• Prozone – Antibody is more than antigen. 
• Post zone – Antibody < antigen.

Also read: How to Prepare Microbiology for FMGE

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