Gram Staining. The Most Commonly Used Differential Stain. Advantages:

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1 Gram Staining The Most Commonly Used Differential Stain Advantages: Can observe size and morphology (like other staining) Can find out additional information about the organism- primarily what type of cell wall/envelope it has This determination leads to more information about the cell, for example, toxin production, enzyme production, characteristic symptoms during infections, types of antibiotics for treatment, and more

2 (A) Gram Negative (B) Gram Positive Cell Membrane

3 Gram Negative Gram Positive

4 Step 1: Start with a smear prep: Think of your smear preps from last lab, Were the smears from broths too sparse? Add more this time if the organisms are too sparse, it is difficult to find them and evaluate them Were the smears from solids too thick? Add less this time if it is too thick, decolorization and interpretation is too challenging You should be able to adjust your smears based on your previous results Try to get the smear evenly distributed The lab book suggests using a positive control (a known Gram positive bacterium) and a negative control (a known Gram negative bacterium) along with what organism you are trying to determine. This increases the amount of time it takes to make a smear prep and does complicate the process. I will suggest some organisms to start with as an alternative. Remember a smear prep always finishes with heat fixation

5 Step 2: Flood your slide with crystal violet (primary stain) All the Gram stains are in the Gram staining kits at each table (there are 3 or 4 per table) Rinse off after one minute (longer than one minute will not cause any problems) Gently tap off the excess water All bacteria are stained purple at this time Step 3: Flood your slide with Gram s iodine (mordant) This forms an insoluable complex with the crystal violet and attaches to the peptidoglycan layer Rinse off after one minute (longer than one minute will not cause any problems) Gently tap off the excess water

6 Next Decolorize with Gram s decolorizer (alcohol/acetone) This is the critical step Hold your slide at an angle and drip decolorizer onto your slide until it runs clear Immediately stop the decolorization by rinsing the slide with water If your smear is uneven, it causes part of the smear to be either over or under decolorized You have to decolorize longer if it is thick, therefore over decolorizing the thinner areas Or if you decolorize the thinner areas perfectly, you may have under decolorized the thicker areas Only YOU can be the judge of YOUR slide, so pay attention to your technique Practice makes you better! Positives are purple and Negatives should be colorless at this step

7 Lastly Flood the smear with safranin (counterstain) Time should be around 1-2 mins (longer will not cause a problem) Rinse off with water and blot in bibulous paper Be sure to rinse stain off the back of the slide as well Observe UNDER 100X WITH OIL IMMERSION This is a DIFFERENTIAL staining procedure you get 2 results: 1) Purple (crystal violet) is POSITIVE 2) Pink (safranin) is NEGATIVE Used slides are put in sanisol dishes, add additional sanisol if needed Close all stain bottles and return to them to the Gram staining kits

8 POSITIVE GRAM STAIN This reaction is associated with bacteria that have a specific cell wall a thick peptidoglycan layer which combines with the crystal violet/iodine complex and is not easily decolorized. Results are best on younger cultures such as 24 hr. cultures When cells die off, they don t pick up the stain as well, such as an older culture Interpret your stain results by considering all of the variables (age of culture, thickness of smear, difficulty of decolorizing, numbers of organisms that appear purple or pink) Sometimes you will be wrong you get better with practice Remember this is a skill these are living organisms some results will not be explained be open minded make your best assessment its simple do you think it is positive or negative?! (Just like Is it a rod or a cocci?) In other words, don t overcomplicate it!

9 Gram Positive Cell Wall

10 Examples of Gram Positives Notice everything is not purple but it is still obvious that the majority is purple Still seeing pink around the edges, that is just safranin collecting or staining cellular or other debris

11 More Examples of Gram Positives: Example of a Gram positive with some variable stained cells Colorless spores inside cells all known spore formers are Gram positive

12 Negative Gram Stain This reaction likely occurs because the peptidoglycan layer is thin and the outer membrane outside of the peptidoglycan layer is easily destroyed by the decolorizer, allowing the crystal violet to be removed easier. The bacterial cells were decolorized (they had been stained with the crystal violet, but it was washed away and they became colorless) and then they were stained with the counterstain, safranin They are now pink Thicker areas may have purple still, some cells may seem purplish, you simply have to decide, based on the thickness of the slide, the difficulty in decolorizing, the age of the culture, etc. As a possible rule it is more likely that Gram positive organisms get over decolorized as a problem, Gram negative organisms are easily decolorized and even if you over decolorize them, the results are still the same they are still pink. If you undercolorize them, they would appear Gram positive. Only YOU can interpret YOUR Gram stain results! You get better with practice!!

13 Gram Negative Cell Wall

14 Examples of Gram Negatives Notice some areas are dark but it s still pink Coccobacilli small rods are common for the Gram negative rods

15 More Examples of Gram Negatives Notice some of the rods look purple but the majority are pink Sometimes they look this light so be careful not to turn your light up too bright

16 Debris and Stain Crystals: notice the size and shapes of the unwanted artifacts