Gram positive and negative bacteria

Methods for making Gram Positive and Negative Controls in Tissue

Gram controls can be made using placental tissue which has been incubated with a heavy suspension of bacteria in a nutrient broth.  Lung may also be used – obtain from autopsy. Culture 24 hours then process.

Specimen

Fresh unfixed placenta or lung – for use on 2nd day of procedure. Gram – positive cocci (Staphylococcus) e.g. Staph. Aureus Gram – negative bacillus e.g. E. coli, Proteus or Klebsiella.

Procedure 1 – Placenta Specimen

1. Inoculate separate blood agar plates with pure cultures of a Gram- positive cocci (Staphylococcus) and Gram-negative bacillus (Proteus or Klebsiella) and incubate for 24 hours at 37°C. (Do not over incubate)

2. The next day the entire inoculum from each plate is suspended in separate 15 mL of Brain Heart Infusion broth (Commercially available).

3. Small 0.5cm clippings of placenta are suspended in the inoculated broth for both negative and positive bacillus and incubated at 37°C for three hours. Placenta is chosen because it is porous and is available, fresh and unfixed, from the labour and delivery rooms of most hospitals.

4. After incubation, the clippings of placenta are fixed in 10% neutral buffered formalin overnight and processed in the usual manner. Once processed embed a small piece of gram positive and gram negative piece side by side in one block.

Procedure 2 – Lung Specimen

1. Obtain cultures containing Gram positive and negative organisms. Spin each type of culture down to concentrate the organisms, remove most of the supernatant.

2. Once the organisms have been resuspended, inject the concentrated culture into a small block of fresh lung. Inject gram positive into one piece of tissue and gram negative into another. Lung tissue works best for this as it has a lot of spaces into which culture can flow.

3. Gently knead the tissue to further distribute the organisms. The lung pieces are then dropped into the fixative.

4. Once processed, embed a small piece of gram positive piece and the gram negative piece side by side in one block.

1. Brown-Hopps method

Reagents – commercial source unknown, unless specified:

Method:

1. Deparaffinize and hydrate to distilled water.
2. Flood the slide with 1% crystal violet solution for 10 seconds.
3. Rinse in tap water.
4. Treat sections with Gram’s iodine for 10 seconds.
5. Wash well in distilled water.
6. Decolourise by gently agitating in acetone, 1 second.
7. Rinse briefly in water.
8. Stain in 1% Basic Fuchsin Pararosaniline chloride for 10 seconds.
9. Wash briefly in tap water.
10. Treat with Gallego’s solution for 10 seconds.
11. Rinse in tap water.
12. Quick rinse in acetone.
13. Rinse briefly with 1% Picric acetone for 1 second.
14. Briefly rinse in acetone.
15. Rinse in acetone/xylene mixture.
16. Clear in 2 changes of xylene and coverslip.

Results:

Gram positive organisms – blue
Gram negative organisms – red
Nuclei – red
Other tissue elements – yellow
*Fibrin, some fungi, Paneth cells granules, keratohyalin, and keratin also stains blue*

2. Gram Microbiological method

Reagents – commercial source unknown, unless specified:

Method:

1. Take sections to water.
2. Crystal violet solution. Filter onto section for 30 seconds.
3. Wash off with Lugol’s iodine.
4. Stain with Lugol’s iodine for 30 seconds.
5. Wash in water.
6. Differentiate in acetone – until dye stops running.
7. Wash in water.
8. Counterstain with dilute Basic Fuchsin for 1 minute.
9. Wash.
10. Dehydrate quickly, clear and mount.

Results:

Gram positive organisms – blue
Gram negative organisms and other tissue structures – red

3. Gram – Twort method

Reagents – commercial source unknown, unless specified:

Method:

1. Deparaffinize and hydrate to distilled water.
2. Flood the slide with crystal violet solution for 1 minute.
3. Rinse in water.
4. Treat sections with iodine for 2 minutes.
5. Wash well in tap water.
6. Rinse sections with acetone until colour stops leeching from them (approx. 2 seconds).
7. Wash well in tap water.
8. Pipette freshly prepared and filtered Twort’s working stain onto slide for 5 minutes.
9. Rinse in alcohol.
10. Clear and mount in DPX-type mountant.

Results:

Gram positive organisms – blue/black
Gram negative organisms – red/pink
Nuclei – red
Red blood cells and most cytoplasmic structures – green
Elastic fibers – black

Disclaimer:

These methods are intended as a guide only. Laboratories that wish to implement these methods should perform internal validation prior to use. The RCPAQAP does not make any claim or warranty for the accuracy or performance of these methods.