Do a lab summary of 300 words minimum on Gram staining.

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Gram stains: see images at the end of this exercise.

The Gram stain is a differential staining procedure developed by Dr. Hans Christian Gram in 1884. The Gram stain differentiates between 2 groups of bacteria: the Gram positives and the Gram negatives. Almost all bacteria can be clearly separated into one of these 2 groups. For this reason, the Gram stain is the first critical step in characterizing and identifying bacterial species. The basis of the method is a difference in cell wall structure, or more accurately, cell envelope structure (includes everything from the cell membrane to the outermost part of the cell). Gram positive cells have a much thicker cell wall which allows these cells to maintain the primary stain (crystal violet) upon being treated with decolorizer solution. The relatively thin cell wall of Gram negative cells loose the purple-violet color of the primary stain when treated with decolorizer. They then take on the red color of the counter-stain (safranin or basic fuschin). Hence, Gram positive cells stain purple and Gram negative cells stain red. Unfortunately, the method doesn't always work perfectly. Several variables can affect the results. Do not try to "cut corners" on the Gram stain. Learn how to conduct the method properly. Practice Gram stains many times before unknown time, otherwise, you could have serious problems identifying your bacterial species.

1) You must conduct Gram stains on "young" cultures. Most bacterial species used in this lab will grow sufficiently to conduct a Gram stain within 18-24 hours given optimum growth conditions (media, temperature, etc.). It is possible that a culture will require more incubation time to grow sufficiently. If so, conduct the Gram stain as soon as growth allows. Why does the age of the culture matter? NOTE: old Gram positive cultures tend to stain Gram negative. This trend is not universal and cannot be depended upon. In general, the older the culture is the less dependable the Gram stain results. The culture will become "Gram variable" which means that some cells will stain purple and others red.

2) Try to conduct Gram stains on cells collected from isolated colonies. This idea applies mainly to bacteria on Petri dishes but may apply to cultures on a slant. Eventhough a culture may appear pure, you cannot be sure. Two neighboring colonies may be different species. Pick one to make sure. If working from a broth, this is impossible. Gram stains should NOT be conducted from broth cultures.

3) In addition to the unknown culture that you stain, you must also stain "control cultures" of Gram positive and Gram negative bacteria. These control cultures must also be young when you stain them. As mentioned above, Gram stains don't always work perfectly even if you adhere to the steps. As your grandmother says, "you have to hold your mouth right." This is why we use controls. Lets say that your unknown culture stains mostly red, but a little purple tint is present. If your Gram negative control looks the same, you can be fairly confident that your unknown in Gram negative. Without a control, you would not have a clue.

4) The importance of a good smear to accurate Gram stain results cannot be over-emphasized.

• NOTE: Thick smears of Gram negative cells are likely to stain Gram positive - Gram variable. If your smear is thick, examine the stained smear in a thinner area.

• Over-mixed smears yield cellular arrangement that is not indicative of the organisms’characteristic cell morphology.

• Staining and examining cells in a smear that was heat fixed while wet is a complete waste of time. Start over.

5) Decolorization is the most critical step in regard to the color accuracy of the Gram stain. You must perfect the method that works best for you and the amount of time to decolorize. This may vary depending on the thickness of the smear, how vigorously you squirt decolorizer on the smear, the angle at which you hold the slide, the time delay between applying decolorizer and rinsing the smear, etc. You should start with 5 seconds and work from there. Over decolorized Gram positives with look Gram negative. Under decolorized Gram negatives will look Gram positive.

6) Some organisms yield inconsistent Gram reactions even if you follow the rules. Examples include species of the Gram(-) genus Neisseria which often stain purple, and an encapsulated organism (we will talk about capsules later) such as Klebsiella pneumoniae. The capsule is a cell covering which may partially prevent the Gram stain reagents from reaching the cell.

Gram stain procedure – I prefer (and we will use) the modified method on the next page.

1) Prepare smears of the unknown culture(s) and the Gram stain control cultures on a single slide, preferably with the unknown(s) between the controls. As I said earlier, I like 3 or 4 smears / slide.

2) Place the slide on the staining tray smear-side up. Cover the smears with crystal violet and allow to sit for 30-60 seconds. Rinse the smears thoroughly but gently with distilled water. The stream of rinse water should not fall directly onto the cells but above them, allowing the water to sheet across the smears. Shake off excess water.

3) Cover the smears with Grams iodine solution (the mordant) for 60 seconds. Rinse as above. Shake off excess water. The mordant helps "lock" the crystal violet into the Gram positive cell wall.

4) CRITICAL STEP: hold the slide at a 45o angle over the sink. Squirt 95% ethanol solution (decolorizer) onto the top edge of the slide so that it sheets down evenly across each of the smears. Depending on the organism and smear thickness, 2-5 seconds of decolorization should be sufficient. Watch the decolorizer as it flows off of the smear. STOP decolorizing immediately when you no longer see color leaching from the smear. Rinse IMMEDIATELY and thoroughly (front & back) as above. Shake off excess water.

5) Lay the slide on the tray. Cover the smears with safranin or basic fuschin (the counterstain) and allow to sit for 60 seconds. Rinse thoroughly as above. Blot dry with bibulous paper. It is microscopy time.

NOTES

* Your success accurately determining Gram stains depends as much or more upon smear quality and

aptitude at microscopy as it does on the Gram stain procedure itself

* Good Gram stains require practice. I would like for you to Gram stain one or more different

organisms each week. This will give you a lot of practice and will help you to become familiar with the

morphology of our organisms.

* Unless I tell you differently, use the Philadelphia Gram stain procedure (next page). It is very good.

* Dr. Gram would appreciate if you capitalize the first letter of his name (Gram).

Philadelphia General Hospital Gram stain modification

1. Prepare smear and heat fix in the normal manner.

2. Add 1-2 drops of crystal violet to smear.

3. Rock back and forth a few times and wait approximately 10 seconds.

4. Add 2-4 drops of 5% sodium bicarbonate to CV on smear to insure (-) charge of Gr (+) envelope.

5. Rock back and forth a few times to mix CV & SB (critical). Wait approximately 10 seconds.

6. Pour CV-SB off slide. Tilt slide over sink and RINSE with a few drops of Graham’s iodine.

7. Add 5-6 drops of Grahams iodine to smear and wait approximately 10 seconds.

8. Pour GI off slide & RINSE with a gentle stream of water.

9. Add acetone drop wise (NOT a stream) to upper end of tilted slide until purple color no longer

comes out of smear – usually takes 2-4 seconds. Watch closely here – easiest step to mess up.

10. Wave slide a few seconds to evaporate the acetone and air dry the smear.

11. Add safranine to cover smear.

12. Rock back and forth a few times and wait approximately 10 seconds.

13. Pour safranine off slide & RINSE with a gentle stream of water.

14. Blot slide dry with bibulous paper and view with microscope.

IMAGES of Gram-stained bacterial cells: under “Laboratory: cellular morphology images”

Gram positive coccus: Staphylococcus: note clusters of round cells

Gram positive coccus: Streptococcus: note chains of round cells

Gram positive rod: Bacillus

Enteric Gram negative rod: Escherichia coli

Non-enteric Gram negative rod: Alcaligenes faecalis