Medical Microbiology [Coagulase Test]

Coagulase test is used to differentiate between Staphylococcus aureus and other coagulase negative species. This test is based on the coagulative properties of S. aureus with plasma. This test is usually performed if the latex agglutination test for S. aureus is invalidated or equivocal.

S. aureus produces 2 forms of coagulase – free and bound. Different types of tests are used to observe the coagulation reactions, depending on the form of coagulase targeted.

Bound coagulase, also known as “clumping factor”, is detected using the slide test. This form of coagulase is bound to the bacterial cell wall, hence its name. It reacts with fibrinogen in plasma to form visible clumps. However this test is not performed in the lab anymore.

The tube test is done on free coagulase. Free coagulase released by the bacterial cell is often found in culture filtrates. It exists as a free form, unlike the bound coagulase attached to the cell. The free coagulase acts on prothrombin, producing a complex with similar properties to thrombin - staphylothrombin. This substance then acts on fibrinogen to form a fibrin clot in plasma.

1. 2-5 staphylococcal colonies are suspended in 0.5ml of rabbit plasma w/EDTA.
2. Incubate aerobically at 35^oC for 4 hours.
3. Check for clot formation.
4. If negative, continue incubation overnight and check again after the extended incubation.

Positive (+ve)	Visible clumps suspended in plasma
Negative	Absence of clumping

NOTE: Care should be taken when examining the clot formation. If too much time has passed before examining the clot formation, it could produce false negative results. This is due to the ability of certain S. aureus to produce staphylokinase, which breaks down any clot that has formed.

References:

"http://www.hardydiagnostics.com/catalog2/hugo/CoaguStaph.htm"

Also taken from http://en.wikipedia.org/wiki/Coagulase on 27 Sep 2009.

EDIT: By the way this entry is from MUNA.
teehee. XD

Fecal Flotation

Hello!!! The test that i am going to talk about today is the fecal flotation test.

A fecal flotation test is normally used to detect the fecally-shed eggs (and sometimes the larvae) of various parasitic worm species as well as the oocysts shed into the droppings by various, replicating protozoan organisms.

The results will help the trainers to decide whether if it is time to deworm the horses.

The equipment that we are using for this test is the Fecalyzer®.

Steps:

• Lift cap and remove green insert. place 1 gram of fecal sample into valve can fill it the FECALYZER® Flotation Medium.
  

• Rotate green insert vial back and forth to separate ova from fecal sample. Mix thoroughly

• Seat green insert vial firmly in place.

• Fill holder completely with additional FECALYZER Flotation Medium to the brim to form a meniscus.

• Cover the float 22mm cover slip on meniscus for 15-20 minutes.
  
  
• Transfer the cover slip to a microscopic glass slide and examine it under the microscope under 40-100x magnification.

Here are some of the pictures or parasite eggs:

• Isospora oocyst (extreme close-up - 1000x) starting to undergo maturation to its infectious state

• A Spirometra tapeworm egg under 1000x oil immersion.

• a Whipworm or Trichuris vulpis egg under 1000x oil immersion.

pictures taken from: http://www.pet-informed-veterinary-advice-online.com/fecal-flotation.html

Regards,

Joash Ong(0702657H)

Medical Microbiology - idenification of bacteria

Hello all, Yan Ling here. First, I'm sorry for the late post, supposed to blog last wk. But here I'm now going to talk abt bacteria identification that I learnt during my 3 wks at microbiology at my company I'm attached to.

But let me explain the type of plates usually used for cultures. For urine culture, sheep blood agar, MacConkey agar, CLED agar and Mellur Hinton(MH) agar with E.coli lawn are used. MH agar with E.coli lawn is used to identify if the patient is taking antibiotics as if there is presence of antibiotics in urine, there will be clearing on the bacteria lawn. For genital swab, eg, high vaginal swab etc, sheep blood agar, MacConkey, GC Lect agar and Sabouraud Dextrose (Sab Dex) agar are used. GC lect agar is used to identify ,Neisseria gonorrhoeae and Sab Dex agar is used for identify presence of fungi at genital site. For wound swap, 2 blood agar and MacConkey agar are used. 2 blood agar are required as one is for aerobic bacteria and another is for anaerobic bacteria. And lastly for ear and eye swabs, blood agar with S. aureus streak, MacConkey and cooked meat medium are used. S.aureus streak is used as they haemolyse the RBC in the blood agar, and thus allowing H. influenzae to grow as H. influenzae requires Cooked meat medium is an enrichment broth which allows growth of any bacteria present in the swab. The sequence of which type of agar to streak first is: blood agar -> macconkey agar -> follow by the required agar for each type specimen. Blood agar is usually streak first as it is a universal agar which allows any type of bacteria to grow. MacConkey agar as you all know, selects only gram negative bacilli to growth and helps to differentiate between lactose fermenters (pink colonies) and non-lactose fermenters (non-pink colonies).

Streptococcus VS Staphylococcus
Streptococcus (gram-positive, catalase negative) appears translucent and shiny on blood agar. There are 3 types of haemolytic strepococcus. Alpha-haemolytic where it is only partial haemolysis, Beta-haemolytic which is complete haemolytic and gramma-haemolytic where there is no haemolysis. In alpha haemolytic, S. pneumoniae is clinically significant, and to identify S.pneumoniae, optocin is used as S.pneumoniae is sensitive to optocin. For beta-haemolytic, there different groups, namely Grp A, B, C, F and G. And for gramma-haemolytic, it is Grp D. The grouping are according to the Lancefield Grouping. Grama-hemolytic are identified as enterococcus sp. which is bile esculin positive which turns bile esculin black.

Staphylcoccis(gram-positive, catalase positive) appears creamy on blood agar and has larger colonies than strepotococcus. Coagulase is used to differentiate S.aureus and other types of staphylcoccus. S.aureus is coagulase positive.

Gram-negative bacilli
Gram-negative bacilli(GNB) can be oxidase positive or oxidase negative. Oxidase negative can be sub-divide into lactose fermenters. Identification of GNB is by using Microbact which is smth to the api 20 that we learnt during mmic pract, but it is used for GNB only. For oxidase negative GNB, only microbact 12A is used and for oxidase positive GNB, microbact 24E or 12A and B are used. Microbact is fairly easy to do, inoculate some colonies into saline to MacFarland 0,5 (remember that time when we all are doing the antibiotic sensitivity test, we use the MacFarland as the guide to how turbid the saline with colonies should be?). Then add 4 to 5 drops to each well of the microbact and add mineral oil to those wells with black rings as mineral oil prevents oxidation. Then it is incubate overnight and colour change is observed and noted down value of colour change using the table they provide. add up the values to form a 4-digit number, then using microbact system, key in the digit and yeah~ we got our bacteria. The system will give a list of bacteria with the probability, only probability of >75% is considered clinically significant. If is < 75%, it will be reported as no pathogen inoculated. Microbact is actually those biochemical tests like triple sugar test, hydrogen sulfate, indole test, urease test etc but in a smaller and more convenient way.

Here is a website that have some information about microbact: http://www.rapidmicrobiology.com/news/603h77.php?s=wells. Retrieved on Sept 13, 2009.

Okay, will say till here, these are the more important ones of bacteria identification. If i explain all, i think the post will be super long.

Feel free to ask any qn~! I will try to answer them. :)

yaNLing xD

Hi. This is Jennifer. Today i will be updating about catalase test which is done to defferentiate to identify organism species.

Catalase Test

Catalase is enzyme which breaks down hydrogen peroxide into oxygen and water. One of the oxidative end products that are produced by aerobic carbohydrate metabolism is hydrogen peroxide. If hydrogen level is too high for bacterial cells, it will kill them.
In this case, the catalase test that is uses to differentiate Neisseria gonorrhoeae and other Neisseria and related Species. ( Kingella denifrificans)
Catalase converts hydrogen peroxide into water and oxygen shown by the following reaction:

catalase
2(H2O2) --------> 2(H20) + O2 (Gas bubbles)


Method
1. A small drop of the hydrogen peroxide solution was dropped onto a plastic surface, eg. Small agar plate or microscope slide.
2. A small amount of cells were transferred from a well- isolated colony from the agar to mix with the solution.


Results
Positive: Explosive bubbling reaction should occur right immediately when the cells and solution were mixed.

Negative: No bubbling reaction.

Below is a picture showing the result of a positive and negative catalase test.
On the left shows a positive catalase test by having immediate explosive bubbling reaction.
On the right shows a negative catalase test by having no bubbling reaction.