Microbiology Bacteriological Techniques

Microbial Sample Sequence

• Collect the sample.
• Perform Gram stain.
• Inoculate culture media.
• Incubate for 18-24 hours at 37°C.
• Check for growth.

Bacteriological Media

• Agar: A semisolid medium.
• Broth: A liquid medium.
• Plate: A flat, round container of agar.
• Tube: A screw-top container, potentially containing agar or broth.
• Slant: A tube of agar that has solidified on a slant.
• Selective Media: Medium containing compounds that inhibit the growth of certain organisms, useful for isolating particular types of bacteria. Examples include MacConkey II Agar, Bismuth Sulfite Agar, Eosin Methylene Blue, Columbia Colistin-nalidixic acid agar (CNA), and Mannitol Salt Agar. Gram-positive or Gram-negative bacteria can be selected for based on the compound.

Differential Media (Examples)

• MacConkey II Agar (Mac): Differentiates lactose fermenters from non-lactose fermenters; neutral red indicator stains lactose fermenting colonies purple. This agar inhibits overgrowth of E. coli vs Klebsiella spp.
• Salmonella-Shigella agar (SS): Selects for pathogenic enteric Gram-negative bacteria, differentiating colonies based on lactose fermentation.
• Mannitol Salt Agar (MSA): Selects for halotolerant organisms (example: Staphylococcus) and differentiates organisms that can ferment mannitol from those that cannot. A lower pH causes phenol red to turn yellow.

Hemolysis

• Hemolysis is the destruction of red blood cells (RBCs), releasing their contents into the surrounding fluid.

Eosin Methylene Blue (EMB) Agar

• Dyes inhibit growth of Gram-positive bacteria.
• Selects for Gram-negative bacteria.
• Differentiates between lactose fermenters and non-lactose fermenters.

Columbia Colistin-nalidixic acid agar (CNA)

• Colistin and nalidixic acid inhibit Gram-negative organisms.

Phenylethyl alcohol (PEA) Agar

• Used for the growth of Gram-positive bacteria.

Mannitol Salt Agar (MSA)

• Contains 7.5% sodium chloride (a high salt concentration), used to select for organisms that are halotolerant (example: Staphylococcus spp.).
• Mannitol and phenol red (a pH indicator) can differentiate organisms that ferment mannitol from those that cannot.

Mueller-Hinton Agar (MH)

• A general-use medium (commonly used in antibiotic sensitivity testing).
• Used in the Kirby-Bauer test and can be enriched with blood for fastidious organisms.
• Measuring zone diameters can help determine an organism's susceptibility.

Salmonella-Shigella Agar (SS)

• Selecting pathogenic enteric Gram-negative bacteria.
• Differentiating colonies based on lactose fermentation (pink colonies)
• Differentiating Hydrogen Sulfide (H2S)-producing bacteria with ferric citrate in the formula (black pigment).

Gram-negative lactose fermenter

• Escherichia coli

Gram-negative, non-lactose fermenter, hydrogen sulfide producer

• Salmonella

Gram-negative, non-lactose fermenter, non-hydrogen sulfide producer

• Shigella

Broths for general use

• Trypticase soy broth (TSB).
• Brain heart infusion (BHI).

BHI use in turbidity

• Brain heart infusion (BHI) broth is used in preparing a bacterial solution at a specific turbidity level.

Enrichment medium

• Brain heart infusion broth, or BHI, can be used as an enrichment medium for growing Salmonella and Shigella when used during fecal culturing.

Urea Broth/Agar

• Determines if bacteria produce the enzyme urease, which degrades urea into ammonia, water, and carbon dioxide.

Bile Esculin Agar (BE)

• Identifying bacteria that hydrolyze esculin (enterococci).
• Positive test produces a dark brown to black colour change.

Simmons Citrate Agar

• Identifies organisms that use citrate as sole carbon source.
• A positive reaction turns the agar a deep blue color.

Triple Sugar Iron Agar (TSIA)

• Determines whether bacteria ferment three types of sugars: glucose, lactose, and sucrose.

Sodium Chloride 0.85% (NaCl 0.85%)

• A sterile solution used for diluting Gram-negative bacteria before testing (API 20E).

Catalase Test

• Detects the enzyme catalase.
• Positive result is indicated by bubbling.
• Examples of catalase-positive bacteria: Staphylococcus. Examples of catalase-negative bacteria: Streptococcus.

Coagulase Test

• Tests for the presence of coagulase enzyme production, differentiating Staphyloccocus species (which can produce the enzyme), from other bacteria. Positive test indicated by a clear gel formation of the sample mixture.

Control Organisms

• Organisms used in lab tests to verify accuracy and reliability.

Optochin Sensitivity Test

• Tests for Streptococcus pneumoniae (a bacterial species). Arrangement of Streptococcus pneumoniae: Diplocci.

Oxidase Test

• Detects cytochrome oxidase.
• Positive result is indicated by a colour change (pink, blue, or yellow).

CAMP Test

• Tests for Streptococcus agalactiae.

Mastitis

• Inflammation of the mammary glands in animals.

California Mastitis Test (CMT)

• Cow-side test for mastitis. Detects somatic cells in the milk.

Common Mastitis Pathogens

• Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, and Escherichia coli.

CMT Positive Next Steps

• Milk culture, BAP, MAC (or specialized mastitis media) and subculturing.