Tilt the slide slightly and gently rinse with tap water or distilled water.Ĭrystal violet is a water-soluble dye which enters the peptidoglycan layer in the bacterial cell wall.Ģ. Gently flood the smear with crystal violet and leave for 1 minute. Gram stain procedure - Gram staining a sampleġ. This kills the microbes in the smear and fixes the sample to the slide, be careful not to overheat the sample however as this can distort cellular morphology. Once the smear has air dried, pass the smeared slide through a flame two or three times. Resuspend a loop of colony material in sterile phosphate buffered saline (PBS) and then proceed as for a liquid culture.ģ. If the source material is from a bacterial plate: For very dense cultures it may be necessary to pre-dilute your culture to ensure individual bacterial cells can be seen under a microscope following staining. Gently smear the droplet in a circular motion into an area of approximately 1 cm diameter. If preparing your slide from a liquid bacterial culture:ĭab a small drop culture onto the slide using a sterile loop. Ensure you use a pencil as many inks are removed by the reagents used in the staining procedure.Ģ. Label a clean glass microscope slide with your sample identification. Gram stain procedure - Preparing a sampleġ. One such useful classification – if a bacterium is Gram positive or Gram negative - is based on the structure of bacterial cell walls. Depending on the characteristic being studied, bacterial species may be broken down into broad groups, but taken together this information can narrow the possible identities greatly. This includes characteristics like their shape (bacilli vs cocci for example), growth in particular nutrients and preference for high or low oxygen environments. But even without getting into the molecular nitty gritty, there are phenotypic differences between groups of bacteria that can be used to differentiate them. Bacterial species, and even specific strains can be differentiated using a number of molecular techniques such as PCR, quantitative PCR, genome sequencing and mass spectrometry. Read more on Gram stain protocol fixes and other common microbiology stain troubleshooting in our ebook, Lab Technician’s Guide to Troubleshooting Common Issues with Biological Stains.Being able to differentiate bacterial species is important for a host of reasons, from diagnosing infection or checking food safety, to identifying which species it is that gives a cheese it’s fantastic character. You will need to adjust the procedure to match your new decolorizing mix.įalse Gram-negative stains can cause a plethora of issues for patients, so if these fixes aren’t working for you, please call the technical help team at 85 or email at If you switch suppliers or make your own decolorizer, the new decolorizer can impact performance. A 50/50 acetone decolorizer will decolorize much quicker than the 25/75 mix.

Examine acetone in your procedure – The main decolorizing factor in your protocol is the acetone.

Read the QC slides – If the Quality Control slide is being over-decolorized, then most likely the specimen is also matching.

25% acetone is the slowest, and 75% acetone is the fastest. The acetone percentage determines the rate of decolorizing.

Change your decolorization length of time – Reduce the time in the solvent and rinse quickly, or use a decolorizing solution that is easier to control for the decolorizing step.

Reduce the time the specimen is exposed to the counterstain to under 30 seconds.

If the specimen is overexposed to the counterstain, however, it can replace the crystal violet-iodine complex in gram-positive cells. Reduce time in counterstain – By rinsing the slide with a counterstain, safranin, all cells are stained red.Open bottles lose more than 90% of available iodine in 30 days, while closed bottles lose about 50% in the same time frame. Keep iodine bottles closed when not in use. Increase iodine exposure – Insufficient iodine exposure due to lower concentration of iodine can affect decolorization.To avoid this issue, don’t use more than a five-second water rinse at any stage of the procedure. Crystal violet is susceptible to washout with water before binding to iodine, creating a false negative test. Reduce water rinse time – Excessive washing can cause excessive decolorization.In order to reduce the risk of speedy decolorization, reduce the heat during fixation.

This can make cells more easily decolorized during the subsequent protocol steps. Excessive heat during the fixation process alters cell morphology and breaks down cell walls. Reduce heat during fixation – Gram staining works by first staining bacterial properties within the cell wall, then removing the excess stain using a decolorizer.Ethos+Bangs Assay & Lateral Flow Development.

Gram positive gram negative bacteria