However, in Gram negative bacteria, the outer membrane is degraded, the thin peptidoglycan layer is unable to retain the crystal violet-iodine complexes and the color is lost.Ĥ. In Gram positive bacteria, the large crystal violet-iodine complexes are then unable to penetrate and escape the thick peptidoglycan layer, resulting in purple stained cells. Immediately rinse with water to avoid over-decolorizing.ĭecolorizer dehydrates the peptidoglycan layer, shrinking and tightening it. Tilt the slide slightly and apply the alcohol drop by drop until the alcohol runs almost clear (5-10 seconds). Decolorize the smear using 95% ethyl alcohol or acetone. Gram's iodine solution (iodine and potassium iodide) is added to form a complex with the crystal violet, which is much larger and is insoluble in water.ģ. Tilt the slide slightly and gently rinse with tap water or distilled water. Gently flood the smear with Gram’s iodine and leave for 1 minute. 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. A copy of the license is included in the section entitled GNU Free Documentation License.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. Permission is granted to copy, distribute and/or modify this document under the terms of the GNU Free Documentation License, Version 1.2 or any later version published by the Free Software Foundation with no Invariant Sections, no Front-Cover Texts, and no Back-Cover Texts. CC BY-SA 3.0 Creative Commons Attribution-Share Alike 3.0 true true share alike – If you remix, transform, or build upon the material, you must distribute your contributions under the same or compatible license as the original.You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. attribution – You must give appropriate credit, provide a link to the license, and indicate if changes were made.to share – to copy, distribute and transmit the work.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |