Week 5: Investigation lab (Microbiology)

Oh my, 5 weeks of our attachment have already passed. I'm have also moved onto another lab. From the central processing area (CPA), I'm now in the investigation lab (IV lab). In CPA, we 'prepare' the patients' specimens for testing. Then, these prepared specimens will be sent to the IV lab so that the bacteria present can be identified, so that a diagnosis can be obtained. Afterwhich the appropriate treatment can be given.

Having said so, it also means that the IV lab plays an important role as to whether the patient will receive the correct treatment. So as interns, we are not given much investigation work to do. Instead, we are being tasked with simpler and 'save-able' jobs. 'Save-able' as in if we do anything wrong, they can repeat the test again.

So far, I have done these few things:

- Subculture cooked meat and brain heart infusion broth (BHIB)

- Perform latex agglutination for the identification of Staphylococcus aureus

- Perform antibiotic susceptibility testing for urine cultures.

Subculture of cooked meat and BHIB

Cooked meat is an enriched media used to cultivate anaerobic bacteria. But according to my mentor there, cooked meat is also used as back-up. In some cases, there may be too little bacteria present in the specimen to show up on the agar plates. As the specimens are submerged into the cooked meat media, any bacteria present cannot 'escape'. After incubating the cooked meat for 1 day at 35°, the media will be subcultured onto blood agar and anaerobic plates. Blood agar is used as most bacteria can grow on blood agar. Anaerobic plates are used to detect the presence of any anaerobic bacteria.

Cooked meat medium [1]

BHIB is a general-purpose medium normally used to cultivate fastidious, non-fastidious, aerobic and anaerobic bacteria. But this BHIB has added antibiotics, oxacillin, to detect methcillin-resistant S. aureus (MRSA). So all bacteria, excluding MRSA, sensitive to oxacillin will be killed. MRSA will not be killed by oxacillin as it is resistant to oxacillin. So this type of BHIB is used for MRSA screening. The BHIB will be incubated for 1 day at 35°, and after 1 day, the broth will also have to be subcultured onto MRSA plates.

MRSA plates are actually made up of either blood agar or mannitol salt agar with added methicillin or oxacillin. It is selective media used to detect MRSA. Presence of MRSA can be indicated by purple colonies present on the agar.

MRSA growing on MRSA plate [2]

Latex agglutination for identification of S. aureus.

This test works by detecting 3 components:
- Fibrinogen affinity factor, also known as bound coagulase or ‘clumping factor’
- Protein A, which has an affinity for crystallisable fragment (Fc) of the gamma

immunoglobulins (IgG)

- Capsular polysaccharides of S. aureus

The reagent used in this test contains latex particles that have been sensitized with fibrinogen, IgG and specific monoclonal antibodies specific against capsular polysaccharides of S. aureus. A combination of these 3 components allows both highly and poorly encapsulated strains of S. aureus to be recognized

For highly encapsulated strains of S. aureus, antibodies specific to the capsular polysaccharide will be used to agglutinate the bacteria. For poorly encapsulated strains of S. aureus, the fibrinogen and IgG will be used to agglutinate the bacteria.

Positive result = agglutination observed with loss of'red background

Negative result = no agglutination with no loss of red background

False-positive results may occur when there is a cross-reaction with Streptococcus spp. as some strains of streptococcus contains a protein with an affinity for the Fc portion of the IgG. So it may react with the latex reagent, thus causing a false positive result.

False-negative results may also occur as some S. aureus do not produce fibrinogen affinity factor (clumping factor), protein A, or capsular polysaccharides. Without the capsular polysaccharides, no specific antibiotics will produced.

Antibiotics susceptibility testing

This form of testing is used as a guide to the doctors as to which type of antibiotics would work best to kill the bacteria present in the patient. So tests are performed in vitro and how well the organism grows in response to the antibiotics will be measured. [6] The antibiotics susceptibility testing is normally done using Mueller-Hinton agar. Mueller-Hinton agar is used for this test because of a few reasons:
- Good reproducibility of the medium
- Simplicity of its formula
- A lot of experimental data are available regarding the usage of this medium [7]

Type of antibiotics to be used for the test is determined by a few factors:
- Target location of the antibiotics (e.g Antibiotics meant to treat bacteria in the brain

must be able to pass through the blood brain barrier)

- Target bacteria, as some antibiotics work better for gram-positive bacteria, while other

antibiotics work better for gram-negative bacteria)
- Avaliability of the antibiotics, as some antibiotics may be more commonly used than others.

As mentioned above, different types of bacteria will require different groups of antibiotics to be used. The groups of antibiotics are described as ‘lines’.

Staphylococcus = A line

Streptoccus = E line

Enterococcus = BE line

Pseudomonas aeruginosa = E line

*Enterobacteriaceae = F line

*Enterbacteriaceae refers to any other bacteria not mentioned in the previous lines. They include Escherichia coli, general Pseudomonas and so on.


Procedures
1. Inoculate single colonies of bacteria into saline. The turbidity of the mixture should match

the 0.5 MacFarland turbidity standard.

2. Dip a cotton swab into the mixture and drain off excess mixture.
3. Swab the entire surface of Mueller-Hinton agar with the cotton swab by turning 60 degrees

after each swab.

4. Stamp the appropriate group of antibiotics onto the Mueller-Hinton agar.
5. Incubate plates at 35° for 1 day.

Result

Bacteria susceptible to the antibiotics = zone of inhibition will be observed around the disk.
Bacteria resistant to the antibiotics = zone of inhibition will be absent around the disk.

Antibiotics susceptibility test [3]

So these are basically what I'm doing in the IV lab. I will be starting on biochemical tests next week. Yes, all the urease, oxidase, catalase tests and so on. So wish me luck!

Picture references:

1. http://www.oxoid.com/uk/blue/press/press.asp?art=Y&arch=Y&pRef=PR023605&c=uk&lang=en&yr=2005

2. http://www.chemoquip.com/

3. www.med.unsw.edu.au/pathology-cds/

-Li Ping-

TG 02