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Tests for Gram Positive Organisms Part II

Experiment 8C lab period 2

We will continue to introduce new tests used for gram-positive organism. Be sure to review results for BAP, MSA, and Coagulase tests from last lab period

Antibiotic Susceptibility Tests

Two separate tests for antibiotic susceptibility will performed. These tests are used to differentiate between alpha and beta hemolytic organisms. Alpha hemolytic organisms (Streptococcus pneumoniae and Streptococcus mitis) are tested for their susceptibility to Taxos P (optochin). The beta hemolytic organisms (Streptococcus pyogenes and Streptococcus agalactiae) are tested with Taxos A (bacitracin). A zone of inhibition of growth around the Taxos disc indicates that the organism is susceptible/sensitive to that antibiotic.

Taxos A (bacitracin sensitivity testing)

This is a differential test used to distinguish between organisms sensitive to the antibiotic bacitracin and those not. Bacitracin is a peptide antibiotic produced by Bacillus subtilis. It inhibits cell wall synthesis and disrupts the cell membrane. This test is commonly used to distinguish between the b-hemolytic streptococci: Streptococcus agalactiae (bacitracin resistant) and Streptococcus pyogenes (bacitracin sensitive). The plate below was streaked with Streptococcus pyogenes; notice the large zone of inhibition surrounding the disk.

Taxos P (optochin sensitivity testing)

This is a differential test used to distinguish between organisms sensitive to the antibiotic optochin and those not. This test is used to distinguish Streptococcus pneumoniae (optochin sensitive) from other a-hemolytic streptococci (optochin resistant).

Procedure:

  1. Obtain a fresh BAP (1 plate per group of 4) and divide it into quadrants. Label the quadrants with the names of the bacteria as shown in the diagram below. Note: To complete this experiment, a stock culture of Streptococcus agalactiae and Streptococcus mitis will be provided. The remaining cultures (Streptococcus pneumoniae, and Streptococcus pyogenes) will be taken from the blood agar plates inoculated in the previous lab.

  2. Using an inoculating loop, pick up a small, isolated colony of Streptococcus pneumoniae from the BAP plate and transfer it to the center of the quadrant labeled Streptococcus pneumoniae. Spread bacteria around in a circle at least the size of a 50¢ piece.

  3. Repeat step 2 with the remaining 3 bacterial isolates. 

  4. Place a taxos P disc (impregnated with optochin) on the Streptococcus pneumoniae and Streptococcus mitis smears.

  5. Put a taxos A disc (impregnated with bacitracin) on the Streptococcus pyogenes and Streptococcus agalactiae smears.Taxos test plate diagram

  6. WITHOUT INVERTING, place the plate in the tray on the side bench. Incubate the plates at 37oC for 24h.

Results

Observe the results for both Taxos A and P. Streptococcus pyogenes will be sensitive to bacitracin and Streptococcus pneumonia will be sensitive to optochin.

Taxos A Strep pyogenes sensistiveTaxos P Strep pneumonia sensitive

Nitrate Test

This is a differential medium. It is used to determine if an organism is capable of reducing nitrate (NO3-) to nitrite (NO2-) or other nitrogenous compounds via the action of the enzyme nitratase (also called nitrate reductase). This test is important in the identification of both gram positive and gram negative species. 
After incubation, these tubes are first inspected for the presence of gas in the Durham tube. In the case of non-fermenters, this is indicative of reduction of nitrate to nitrogen gas. However, in many cases gas is produced by fermentation and further testing is necessary to determine if reduction of nitrate has occurred. This further testing includes the addition of sulfanilic acid (often called nitrate I) and dimethyl-alpha-napthalamine (nitrate II). If nitrite is present in the media, then it will react with nitrate I and nitrate II to form a red compound. This is considered a positive result. If no red color forms upon addition of nitrate I and II, this indicates that either the NO3- has not been converted to NO2- (a negative result), or that NO3- was converted to NO2- and then immediately reduced to some other, undetectable form of nitrogen (also a positive result). In order to determine which of the preceding is the case, elemental zinc is added to the broth. Zinc will convert any remaining NO3- to NO2- thus allowing nitrate I and nitrate II to react with the NO2- and form the red pigment (a verified negative result). If no color change occurs upon addition of zinc then this means that the NO3- was converted to NO2- and then was converted to some other undetectable form of nitrogen (a positive result).

If the nitrate broth turns red (tubes pictured in the center) after nitrate I and nitrate II are added, this color indicates a positive result. If instead, the tube turns red (tube pictured on the left) after the addition of Zn, this indicates a negative result. If there is no color change in the tube after the addition of nitrate I and nitrate II, the result is uncertain. If the tube is colorless (picture on the right) after the addition of Zn this indicates a positive test.

In today's lab we will begin the characterization of Gram-negative bacilli. The nitrate test, which is important for the identification of both Gram-positive and Gram-negative bacteria, will be performed.

Innoculation Procedure

Results procedure

Additional steps are required to complete the nitrate test. There are two ways to read a positive result but only one way to be negative. 

Results

(From left to right) Streptococcus pneumoniae- Turned red after Zn; Staphylococcus epidermidis & Escherichia coli- Turned red after Nitrate I and II; Pseudomonas aeruginosa- Stayed colorless after Zn 

Example of each possible nitrate results

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