Changes to MI 822 Lab 2

Sept. 6, 2000

 

 

Most of the Demonstrations and Student Work sections in Lab 2 of the MI 822 lab manual remain the same except for the changes in the following sections:

 

            Demonstration A-2c (pp. 2-6 to 2-7)

            Demonstration/Student Work B-2d (pp. 2-10 to 2-11)

            Student Work B-2e (pp. 2-11 to 2-12)

            Unknown #1 (pp. 2-15 to 2-16)

 

The changes for each section are discussed below.

 

 

Demonstration A-2c  2.) Rapid diagnostic test for S. aureus, done with Staphyloslide.  (pp. 2-6 to 2-7)

 

            The diagnostic kit chosen for this experiment is the BactiTM Staph (Remel, Lenexa, KS).  The protein-coated latex particles are capable of detecting both clumping factor and protein A.  Similar to Staphyloslide test, adherence of S. aureus to the black latex particles causes visible agglutination.  S. epidermidis and S. saprophyticus should not cause agglutination; however, heavy inoculation with S. saprophyticus has produced false positives with this test.

 

An instructor at each station will demonstrate the BactiTM Staph test using cells of S. aureus and S. epidermidis.

 

Procedure:

 

1.      Gently mix the Staph latex reagent bottle (make sure latex is resuspended and warmed to room temperature) and place 1 drop of the latex reagent (by holding dropper bottle vertically) into one circle of the reaction card.  Repeat this step for each Staphylococcus sp. tested.

2.      Spread 1 colony (using either an applicator stick or an inoculating loop) onto the circle and then mix into the drop of latex reagent.  Slowly blend the staphylococci into the reagent spreading the mixture over much of the circle.  Discard the stick into biohazard waste or flame the loop.

3.      Rotate the slide with circular motion for up to 60 sec.  Aggregation of the black latex suspension with subsequent loss of black background represents a positive reaction for agglutination.  For S. aureus, this usually occurs within 15 sec.  A negative reaction is reported as little or no agglutination (without the loss of black background) within 60 sec.

4.      It is important to test the latex reagent for autoagglutination as well as for performance with BactiTM Staph Positive and Negative Controls on a daily basis to validate subsequent test results.

 

Limitations of the Procedure:

            Some Staphylococcus sp. (including S. saprophyticus) may cause false positive results.  A lighter inoculum usually solves this problem.

            Rough or stringy colonies may produce aberrant reactions.  Other organisms (particularly yeast) may cause agglutination.  A Gram stain should be performed prior to the test so that only staphylococci are tested.  If no interpretation can be determined from reactions of the latex reagent with a light and heavy inoculum, then identification should be based on the coagulase tube method or other biochemical tests.

 

 

Demonstration/Student Work B-2d. Latex Agglutination Test for Streptococcal Group Identification: (pg. 2-10 to 2-11)

 

            The latex agglutination test used for this section is the PathoDx Strep Grouping kit (Remel, Lenexa, KS).  Similar to Streptex (Murex/Abbott), the Strep Grouping Kit identifies beta-hemolytic streptococci of Lancefield Groups A, B, C, F, and G.  The group-specific carbohydrate antigen is extracted from the cell envelopes of the isolated Streptococcus sp. by a nitrous acid extraction at room temperature.  The mixture is then neutralized and added to the latex particles coated with IgG to the Group-specific antigen. According to the technical literature, cross-reactivity from other streptococci should be minimal due to the high specificity of IgG for each streptococcal group antigen and the nature of the nitrous acid extraction procedure.  A separate kit (PathoDx Strep D) is needed to identify the Group D streptococci and enterococci associated with human infections. In this test (described on page Lab 2A-8), isolated colonies are tested directly with latex particles coated with IgG to glycerol teichoic acid (the Group D antigen found in cell walls).

 

            In this particular experiment, students will divide into groups of 4 and one student will perform the PathoDx Group B test on S. agalacticae, a streptococcal strain (GBS) that causes serious neonatal infections.

 

Procedure: (Reagents 1, 2, and 3 must be at room temperature.  It is not necessary for the latex reagents to be at room temperature.  Prior to each lab session, controls will be tested for each latex reagent—in clinical diagnostic laboratories, these controls are tested on a daily basis. The following procedure can be used for both Group A and Group B tests to identify Unknown #1 cultures.)

 

1.      Label one 12 x 75 mm test tube for each sample.

2.      Add 2 drops of Reagent 1 to each tube.

3.      Add 2 drops of Reagent 2 to each tube.

4.      Pick 1 to 4 isolated colonies with a applicator stick or an inoculating loop and mix the colonies with the extraction reagents in the test tube by rubbing the stick or loop against the bottom of the tube.  Discard the stick into the biohazard waste or flame the loop.

5.      Add 4 drops of Reagent 3 to each tube.  Mix by tapping the tube.

6.      Add 1 drop of the extraction (from a transfer pipet) to one end of the oval on the reaction card.  Add 1 drop of Group B (or Group A) latex reagent to other end of oval (before adding latex reagent, resuspend latex by inverting the dropper).  Mix the latex and extract together using a stirrer.  Discard stirrer in biohazard waste.

7.      Rock the card back and forth for no more than 1 minute.  Examine the slide for agglutination.  A positive reaction is agglutination of the blue latex with a loss of the blue background.  A negative reaction remains as a uniform blue milky appearance.

 

Limitations to the Procedure:

            Cross-reactivity or non-specific clumping can occur with large inocula, therefore, use only 1 to 4 colonies in the extraction step.  Also some mucoid strains may cause non-specific clumping of the latex.  Cross-reactivity can occur with Listeria monocyotogenes and large inocula of Protein A-bearing strains of Staphylococcus aureus.  A catalase test should differentiate between Listeria or S. aureus and streptococci.

 

 

Student Work B-2e: Throat swab for the detection of Group A Streptococcus. (pp. 2-11 to 2-12)

 

            A copy of the Technical Information for the RIM A.R.C. Strep A test (Remel, Lenexa, KS) is shown below.  Read the “Test Procedure” and examine the diagrams accompanying the procedure.

 

            Divide into groups of 4 (each group should have 2 tests).  One test will be used with a pure culture of S. pyogenes and the other will be used with a throat specimen.  Both tests will follow the “Test Procedure” and diagrams below.  One student will use a swab to pick one colony of S. pyogenes from BAP and that swab will be used in Step 2.  A second student will swab the posterior pharynx (over tonsillar area) of one student in the group and use that swab in Step 2.

 

For purposes of culturing a throat specimen, a third student will swab the posterior pharynx (over the tonsillar area) of another student in the group and inoculate this swab onto a BAP according to the procedure on page 2-12 of the lab manual (next to the diagram of a tongue).  Don’t forget to stab the agar!!

Note: For both throat swabs, do not touch the swab to the tongue, the lateral walls of the buccal cavity, or the uvula.



Unknown #1: (pp. 2-15 to 2-16)

 


Add to the list of Media and Tests for use in identifying Unknown No. 1, novobiocin and BHI with 6.5% NaCl (don’t forget about the bile esculin slants on the top of page 2-16).

 

Under Step 5 of Procedure for Identifying Unknown No. 1, you will perform a BactiTM Staph or PathoDx Strep test based on the results from the catalase test.   The procedures for BactiTM Staph and PathoDx Strep A and B are given above.  If your tests indicate a Streptococcus sp. and the test for PathoDx Strep A and B are negative, then perform the test for PathoDx Strep D as described below.

 

PathoDx Strep D Procedure (Direct colony testing): (all reagents must be warmed to room temperature.  Controls will be tested prior to the lab session).

 

1.      Place 2 drops of Strep D latex reagent in one oval of the reaction card and 2 drops of the Negative Control latex in a second oval on the card.

2.      Pick no more than 3 to 5 colonies (using a applicator stick or inoculating loop), spread onto the first oval, and mix the Strep D latex reagent with the smeared colonies (Discard stick in biohazard waste or flame loop).

3.      Pick another set of 3 to 5 colonies (using a applicator stick or inoculating loop), spread colonies on the second oval, and mix the Negative Control latex with the smeared colonies (Discard stick in biohazard waste or flame loop).

4.      Rock the card for 1 to 3 minutes.  Check for agglutination.  A positive reaction is distinct clumping of the Strep D latex that is not observed with the Negative Control latex.  A negative reaction is the homogenous suspension of the Strep D and Negative Control latex particles.  A noninterpretable result occurs when clumping of both the Strep D and Negative Control latex particles is observed.

 

Limitations to the Procedure:

 

            False positives or non-specific clumping can occur if the inoculum is too heavy (especially with Protein A-bearing strains of S. aureus) or the strain is mucoid.  A Gram stain and catalase test should give some indications that the colony or strain tested in this experiment is a Streptococcus sp.