Exercise 36: Identifying Unknown Bacteria - Comprehensive Notes

Exercise 36: Identifying Unknown Bacteria

Overview

• The exercise involves identifying two genera of bacteria from a mixed broth: one Gram-positive (G+) and one Gram-negative (G-).

Gram-Positive Bacteria

• Bacillus subtilis: Rod-shaped.

• Cocci in Clusters:

  • Staphylococcus aureus

  • Staphylococcus epidermidis

• Cocci in Chains:

  • Streptococcus pyogenes

  • Streptococcus agalactiae

  • Streptococcus mitis

  • Enterococcus faecalis

Gram-Negative Bacteria

• Serratia marcescens: Produces a water-insoluble red pigment.

• Lactose Fermenting:

  • Escherichia coli

  • Enterobacter cloacae

  • Klebsiella pneumoniae

• Lactose Non-Fermenting:

  • Pseudomonas aeruginosa

  • Proteus vulgaris

  • Salmonella typhimurium

Procedure

Initial Streaking

• Streak the mixed broth culture for isolation in duplicate on:

  • MacConkey

  • TSA (Tryptic Soy Agar)

  • CNA (Colistin and Nalidixic Acid Agar)

• Incubate one set (MacConkey, TSA, CNA) at 37°C and the other set (MacConkey, TSA) at 30°C for 24 to 48 hours.

MacConkey Plate Observation

• Observe the MacConkey plate for growth and colony colors.

• Select a pure colony and streak for purification on two TSA slants.

  • MacConkey #1: Working stock

  • MacConkey #2: Reserve stock

• Incubate the two TSA slants at the optimum temperature determined.

TSA Plate Observation

• Observe the TSA plate.

• Select two different-appearing colonies with different growth characteristics.

  • Use "L" for the larger colony.

  • Use "S" for the smaller colony.

Purification Streaks from TSA Colonies

• Pick from the "L" colony and streak for purification on two TSA slants.

  • L #3: Working stock

  • L #4: Reserve stock

• Pick from the "S" colony and streak for purification on two TSA slants.

  • S #5: Working stock

  • S #6: Reserve stock

CNA Plate Observation

• Observe the CNA plate for growth.

• Select a pure colony and streak for purification on two TSA slants.

  • CNA #7: Working stock

  • CNA #8: Reserve stock

Incubation and Storage

• Incubate all the TSA slants at the optimum temperature determined for each bacterium for 24 to 48 hours.

• Keep the reserve stock cultures (TSA slants #2, 4, 6, 8) refrigerated.

• Refrigerate the working stock cultures (TSA slants #1, 3, 5, 7) between uses.

Gram Staining

• Gram-stain the working stock of each organism.

  • TSA #1 is expected to be G-.

  • TSA #7 is expected to be G+.

  • TSA #3 and #5: One is expected to be G-, and the other G+.

Identifying Gram-Positive Bacteria

Confirmation

• Confirm G+ bacterium: Gram-stain tubes #3, 5, 7.

• Confirm cocci in chains (Strep) or in clusters (Staph) by catalase test in addition to Gram-stain.

Staphylococci

• If cocci in clusters (Gram stain) and bubbles (catalase test) are observed: Perform Blood agar and Mannitol salt agar tests (refer to ex. 33-4) to distinguish between Staphylococcus aureus and Staphylococcus epidermidis.

Streptococci

• If cocci in chains (Gram stain) and no bubbles (catalase test) are observed:

  • Bacitracin on a blood agar: refer to ex. 34-6

  • CAMP test: refer to ex. 34-7

  • Bile esculin: refer to ex. 34-7

  • Salt tolerance (NaCl broth): refer to ex. 34-8

• Use the following table to distinguish between Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, and Streptococcus mitis:

Identifying Gram-Negative Bacteria

• Serratia marcescens: Produces a water-insoluble red pigment.

  • Observe the red pigment of colonies grown on TSA plates and slants.

  • Confirm with the gelatin test (refer to lab exercise 24).

• Colored colonies on MacConkey: use TSA #1. Do IMViC tests (Exercise 29).

  • Lactose fermenting:

    • Escherichia coli: I (+), M (+), V (-), C (-)

    • Enterobacter cloacae: I (-), M (-), V (+), C (+)

    • Klebsiella pneumoniae: I (-), M (+), V (-), C (+)

• Colorless colonies on MacConkey: use TSA #1.

  • Lactose non-fermenting:

    • Pseudomonas aeruginosa

    • Proteus vulgaris

    • Salmonella typhimurium

• Perform the following tests:

  • Carbohydrate fermentation – exercise 25

  • Hydrogen sulfide (SIM medium) – exercise 26

  • Urea hydrolysis – exercise 27

  • Nitrate Reduction – exercise 28

• If you observe a black precipitate in SIM medium and a negative urea hydrolysis: the bacterium is Salmonella typhimurium.

• If you observe a black precipitate in SIM medium and a positive urea hydrolysis: the bacterium is Proteus vulgaris.

• If you observe a negative fermentation and a complete nitrate reduction: the bacterium is Pseudomonas aeruginosa.

Final Step

Name the two identified bacteria.