Observing Aseptic Procedures Throughout This Lab

Question 1

How to identify if a medically gram-positive Cocci: Staphylococcus or streptococcus?

Answer 1

Take your sterile inoculating needle and obtain bacteria from a well-isolated colony(don’t cut the agar ) (catalase )

Palace them on the clean in one spot (do not spread the colony over a large area on the slide)

Place a drop of hydrogen peroxide on the bacteria. Observe for vigorous gas production

Staphylococcus gives a positive result

If you got a positive result, move on to coagulate test

Question 2

Describe the coagulate test

Answer 2

To identify S. Aureus

This test is used to distinguish between different Staphylococci

Using a clean slide:

Place a drop of rabbit plasma on the slide

Remove bacteria from an isolated colony on your agar plate with your sterile inoculating needle

Mix the bacteria in the plasma

Incubate at room temperature with gentle rocking for at least 10 mins

S. Aureus will give a positive result I.e. clotting the plasma

Question 3

If there is a positive on the coagulate test, how do you test to know if you have S. Aureus?

Answer 3

BactiStaph(remel; to identify s aureus)
-Resuspend the kit reagent by gently inverting for 5 seconds

• place a drop in a circle on the slide for each organism
• take a wooden applicator stick and pick 2-5 colonies of identical morphology
• spread the bacteria on the surface of the circle next to the reagent drop.
• mix thoroughly the reagent and the bacteria
• rotate the slide for up to 60 seconds and watch for the appearance of black clumps against a blue background, a positive test
• ignore clumps that form after the limit

Question 4

What is novobiocin resistance?

Answer 4

Using a plate with isolated colonies of either S. Saprophyticus or S. Epidermids

• take a single colony with your sterile inoculating loop and spread it on a TSA plate using the “lawn” method
• Aseptically place a novobiocin disk in the middle of the plate and incubate overnight
• if there is no zone of inhibition (I.e. the bacteria are not sensitive to the novobiocin) it is S. Saprophyticus

Question 5

Plate all staphylococcus on

Answer 5

MSA plates

Question 6

What to do if you have a negative catalase test, what to do?

Answer 6

Move on to PYR, LAP, ESC test

Using an isolated colonies that are potentially S. Pyogenes or other streptococcus
-label the test cards appropriately to identify the specimens with one specimen per card

   - Add 1 drop of rehydrating fluid to each circle on the card
  - inoculate each circle with several identical isolated colonies. Use a wooden applicator stick.

  - incubate at room temperature for 10 mins 
  - add a drop of developer solution to only the PYR and LAP circles

Question 7

What are the results of PYR, LAP, ESC test?

Answer 7

ESC- color change to dark brown or brownish-black

PYR and LAP- if a pink to red color develops within one minute the test is /are positive

Finally: observe hemolysis pattern on the BAP to confirm the streptococcus species

Question 8

Explain latex agglutination

Answer 8

Pick 4 isolated colonies with a disposable applicator stick or an inoculating loop

Rub the colonies thoroughly and smoothly onto the PathoDx slide in the center of the delineated oval

Repeat for each group reagent to be used
-add 1 drop of step A latex to the first oval, 1 drop of strep B latex to the second oval. Continue in the same manner, adding strep C, F and G latex to the remaining three ovals

• mix the latex and the smeared colonies thoroughly with a stirrer, using a clean end for each oval
• A positive agglutination reaction with one of the latex reagents usually occurs in 15 to 60 seconds
• Stop rocking the slide as soon as aa clearly discernible positive reaction is observed and record the result. Do not rock the slide for more than 60 seconds

Question 9

If coagulate is negative…

Answer 9

Then plate on TSA plate