Bacteriological and Mycological Tests

• The notes cover various bacteriological and mycological tests and procedures.
• A Bunsen burner or alcohol lamp should be used for sterile work.

Catalase Test

• Detects the presence of catalase enzyme in bacteria.
• Add 1-2 drops of 3% hydrogen peroxide onto a glass slide.
• Sterilize the inoculating loop and let it cool; get inoculum and smear onto the slide.
• Observe for bubbles: present indicates positive (Staphylococcus spp.), absent indicates negative (Streptococcus spp.).

MRVP Test

• Methyl Red (MR) and Voges-Proskauer (VP) tests go together, using MRVP broth.

Methyl Red (MR) Test

• Detects mixed acid fermentation via acid production.
• Sterilize the inoculating needle and let it cool; stab inoculum into the medium.
• Incubate at 35°C for 24 hours, then add 5 drops of methyl red indicator.
• A red color indicates a positive MR test (e.g., Escherichia coli), a yellow color indicates a negative result.

Voges-Proskauer (VP) Test

• Detects acetoin production via glucose fermentation.
• Sterilize the inoculating needle and let it cool; stab inoculum into the medium.
• Incubate at 35°C for 24 hours, then add 5 drops of Kovac's reagent.
• Shake, and observe reaction up to 30 minutes: a red color is positive (e.g., Enterobacter spp.), no color change is negative.

Triple Sugar Iron (TSI) Test

• Assesses the bacterium's ability to ferment sugars and produce hydrogen sulfide (H2S).
• Uses a medium containing lactose, sucrose, glucose, and ferrous sulfate with ferrous sulfate, which is essential for differentiating enteric bacteria.
• Sterilize the inoculating needle and let it cool; stab the butt of TSI agar slant, retract, then streak the slant surface.
• Incubate at 35°C for 24 hours.
• Observe the reactions:
• Yellow butt and slant means glucose, lactose, and/or sucrose fermentation.
• A black precipitate in the butt indicates hydrogen sulfide production.
• Cracks or lifting of agar indicates gas production.

Simmon's Citrate Agar (SCA) Test

• Determines if a bacterium utilizes citrate as a sole carbon source.
• Sterilize the inoculating needle and let it cool; stab the butt, retract, then streak the slant surface.
• Incubate at 35°C for 24 hours.
• A blue color indicates a positive citrate test (Klebsiella), and a green color indicates a negative test (Escherichia coli).

Sulfur Indole Motility (SIM) Test

• Detects hydrogen sulfide production, indole formation, and motility.
• Sterilize the inoculating needle and let it cool; stab inoculum into the medium.
• Incubate at 35°C for 24 hours.
• Observe reactions: blackening of the medium indicates hydrogen sulfide production (e.g., Proteus).
• Add Kovac's reagent, indicated by a red color on the surface. This means is a positive indole test (e.g., Escherichia coli).
• Diffuse growth (spreading) indicates motility; negative growth only along the stab line.

Quantitative Plating Method

• Involves diluting a sample, spreading it on a solid agar plate, and counting colonies.
• Prepare serial dilutions and label them (e.g., 101, 102, 103)
• Use a micropipette to draw the bacterial sample and transfer it to the first test tube (101)
• Mix thoroughly.
• Take 100 µL from the first test tube and transfer it into the agar plate.
• Repeat for all dilutions, then incubate the plates at the optimal temperature for 24–48 hours, depending on the bacterial species.
• Count colonies using a counter to calculate colony-forming units per milliliter (CFU/mL) with the provided formula, where the numerator equals Number of Colonies times Dilution Factor, divided by Volume Plated (mL).

Mice Inoculation

• Properly restrain the mouse by scruffing the neck and securing the tail.
• Prepare a 1 cc syringe and NSS, ensuring no air bubbles.
• Clean injection site with alcohol, confirm the needle is beveled up, confirm the mouse is properly restrained, and inject it.
• Intraperitoneal injection: insert into the peritoneal cavity, aspirate before injecting; if blood appears, reposition.
• Subcutaneous injection: insert under the skin in the scruff of the neck.
• Aspirate first to ensure you have not hit a blood vessel before proceeding.
• Avoid vital organs, look for blood vessels.
• Remove the needle smoothly and apply gentle pressure to the injection site.
• Other routes: ocular, intranasal, oral.
• Monitor for adverse reactions and dispose of materials properly.

Embryonated Egg Inoculation

• Candle the egg, locate the injection site, clean the surface, then gently inject it.
• Chorioallantoic Membrane (CAM) Route (for Poxvirus)
• Amniotic and Allantoic Routes (for Influenza)
• Yolk Sac Route (for Rickettsiae)
• Seal, incubate, monitor, and observe.

Introduction to Mycology

• Prepare SDA plates, an inoculating loop, a Bunsen burner, and the fungal sample.
• Draw a "Y" on the agar plate as a guide for streaking.
• Dip a cotton swab into the test tube containing NSS, then set it aside.
• Collect the fungal sample and streak it onto the SDA plate.
• Label the plate, place it upside down, and incubate at 25°C to 30°C.
• After 24 hours, check for growth; if none, continue incubating and check again the next day, and document.
• Yeasts are defined as smooth, moist, and creamy colonies (e.g., Candida).
• Molds are defined as fuzzy or powdery colonies of various colors (e.g., Aspergillus).
• Dimorphic Fungi are yeast-like at 37°C, mold-like at 25-30°C (e.g., Histoplasma).
• Filamentous Fungi are long, thread-like growth with branching hyphae (e.g., Rhizopus).
• Spherules and Endospores are round fungal structures containing spores (e.g., Coccidioides).