Week 10

Hi everyone, it’s my turn again this week. For this week, I think I’m going to share about what I’ve learned from the microbiology section from my lab. I’m going to introduce this kit that can help to identify the different types of gram-negative bacteria. It is named the Microbact Gram-Negative Identification System.

Principle:
The Microbact Gram-negative system is a standardized micro-substrate system designed to stimulate conventional biochemical substrates used for identification of Enterobacteriaceae and common miscellaneous Gram-negative bacilli. Organism identification is based on pH change and substrate utilizations.

This product consists of two separate substrate strips, 12A and 12B. Each strip consists of twelve different biochemical substrate. The 12A strip can be used alone to identify oxidase-negative, nitrate positive glucose fermenters and can be used for screening pathogenic Enterobacteriaceae from enteric and urine specimens or identification of other common isolates. The 12B strip can be used together with the 12A strip for identification of oxidase positive, nitrate-negative, glucose non-fermenters and the Enterobacteriaceae.

Each kit contains one holding tray for the 12A and 12B strips and organism ID report forms including colour interpretation chart.

Examples of species of oxidase negative, Gram-negative bacilli that can be identified using 12A strip alone:
Acinetobacter spp., Shigella spp., Enterobacter spp., Escherichia spp., Salmonella spp., etc.

Examples of species of Gram-positive bacteria that can be identified by using combination of 12A and 12B strips:
Vibrio spp., Pseudomonas spp., Pasteurella., etc.

Set up procedure:
1.Isolation

To use the kit for identification of organism, a pure culture of that particular organism must be obtained. Appropriate agar media such as MacConkey, Blood or Chocolate may be used to grow the organism for approximately 18 to 24 hour. I believed you all would know how to do a pure culture plate by now so I won’t state the procedures here.

Before using the kit, an Oxidase test should be performed on that organism to be identified. As mentioned above, if it’s positive, 12A and 12B strips should be used and if it’s negative, 12A strips can be used alone.

2.Preparation of inoculum

Pick 1-3 isolated pure colonies from the 18-24hr culture and emulsify in 2.5 to 5 ml of sterile saline. Usually 1 colony is enough to emulsify in 2.5ml of saline if doing on the 12A strip alone. If it’s doing 12A and 12B strips, it may need 2 colonies to emulsify in 5ml of the saline. Then, mix thoroughly for a uniform suspension.

3.Inoculation

As the strips are sealed, the individual substrate sets can be exposed by cutting the sealing strip and slowly peel it back. Then, place the strip on the holding tray and using a sterile dropper, fill the bacterial suspension to half of each well in the set. After that, drop one drop of sterile mineral oil on the wells that have the substrates underlined (for 12A strip is well 1,2 and 3 while for 12B strip is well 8 and 12).


In case you all want to ask me what each well contains, here’s the answer:
For 12A strips-Well 1- Lysine
Well 2- Ornithine
Well 3- H2S
Well 4- Glucose
Well 5- Mannitol
Well 6- Xylose
Well 7- ONPG
Well 8- Indole
Well 9- Urease
Well 10- VP
Well 11- Citrate
Well 12- TDA

For 12B strips-Well 1- Gelatin
Well 2- Malonate
Well 3- Inositol
Well 4- Sorbitol
Well 5- Rhamnose
Well 6- Sucrose
Well 7- Lactose
Well 8- Arabinose
Well 9- Adonitol
Well 10- Raffinose
Well 11- Salicin
Well 12- Arginine

4.Incubation

Reseal the inoculated rows with adhesive seal and write the specimen identification no.(in my lab case is using barcode no.) on the seal with a marker. Then, incubate 33-37°C for 18 to 24hrs.

5.Reading the test strips

Remove the test strips from the incubator and unseal them for reading. Positive and negative reaction results are recorded down on the organism ID report forms by comparing the colour change with the colour chart. For some of the wells, there’s an additional reagent to add to before reading. For 12A strips, well 8 drop 2 drops of Indole (Kovacs) reagent and read within 2 minutes, well 10 add 1 drop of VPI and 1 drop of VPII and read within 15 to 30 minutes and well 12 add 1 drop of TDA reagent and read immediately. These additional reagents are required for the reaction to start as different bacteria can have different reaction to these reagents.

There’s also an additional test called the Nitrate Reduction Test. It is performed in well 7 (ONPG) after reading the ONPG reaction. 1 drop of Nitrate reagent A and 1 drop of Nitrate reagent B is added to the well and a change to colour red within few minutes indicates nitrate reduction to nitrite. The positive reaction indicates that it is an organism belonging to the family of Enterobacteriaceae.

6.Interpretation

For the interpretation is pretty easy as they adopted a coding system/program that interpret the type of bacteria simple by entering the recorded result. For every 3 reaction (every three wells), it is added up to a single digit of the code. So if only 12A strip is used, there will be 4 digits which make up the code. If using 12A and 12B strips together, there will be a total of 8 digits that make up the code. Simply just choose the correct program and enter the code and the system will tell you what kind of bacteria is it.


Alright, that's it for this week. Enjoy your life people, if you still can now. Lol.

Sharon
Tg01