MUCLecture_2021_111831460.ppt
- 1. Medical microbiology Lab 2: Examination of stained microorganisms: smear preparation and simple staining, Gram staining Asst. Lec. Mariam A. Ali Asst. Lec. Iman. k. kadhim Al-Mustaqbal University college 2 st Class, 1 st Semester Department of pharmacy
- 2. 1-Inoculation: Producing a pure culture 2-Isolation: Colony on media, one kind of microbe, pure culture 3-Incubation: growing microbes under proper conditions 4-Inspection: Observation of characteristics (data) 5-Identification: use of data, correlation, to identify organism to exact species
- 3. 1-Inoculation: Producing a pure culture Introduce bacteria into a growth medium using “aseptic” technique to prevent contamination. Tools: Bunsen burner, loop. Needle, etc. In working with microorganisms, we must have a method of transferring growing organisms from a pure culture to a sterile medium without introducing any unwanted outside contaminants. This method of preventing unwanted microorganisms from gaining access is termed aseptic technique.
- 4. 2-Isolation: Colony on media, one kind of microbe, pure culture: isolation on general and special “differential media” General growth media: NA, TSA Differential: Mac, EMB, SS , These have dyes, salts, inhibiting agents. Gram – bacilli, green sheen on EMB: E.coli
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- 6. • 3- Incubation: Allow organisms to grow under the optimal conditions • Temperature, with or without oxygen etc.
- 7. • 4- Inspection: Observation, description • Colony Morphology, Microscopic examination (grams stain) • Systematic recording of “DATA”
- 8. • 5-Identification: Correlating data from all observations to identify organism to species • Ex., green sheen on EMB, • ferments lactose, Gram – bacilli, • :E.coli. Gram + bacilli Gram - bacilli
- 9. Staining bacteria cells Staining bacteria cells for microscopic examination makes it possible • to define their cell size, shape, arrangement; • to study their chemical properties,and structures. These characteristics can be use for bacterial identification
- 10. Overview of a bacterial staining procedure
- 11. Simple stains use a single basic dye (e.g. crystal violet, methylene blue, safranin) to color bacterial cells so that their size, shape and arrangement can be observed Staining bacteria cells:simple staining one dye
- 12. •Differential stains, such as the Gram stain and the acid-fast stain, differentiate bacteria based on the chemical composition of their cell wall. •Differential stain use two dyes instead of one: the first stain is the primary stain, the second is the counterstain. •A decolorization step occurs between the application of the primary stain and counterstain. •Depending on the composition of the cell wall, bacteria will either retain the primary stain during decolorization or lose the primary stain and take up the counterstain. Staining bacteria cells: differential stain two dyes complex procedure, see difference between cells
- 13. GRAM STAIN PROCEDURE 1. Stain with crystal violet 2%…… ……….….1 min. 2. Gram’s iodine (Lugol)………………………1 min. 3. Wash off with tap wate. 4. Decolorizer (Alcohol 50%-Acetone 50%)…20 sec. 5. Wash off with tap water 6. Safranin 0,25%………………………………1 min. 7. Wash off with tap water 8. Blot dry with bibulous paper •Hans Christian Gram was a Danish bacteriologist. He developed the Gram stain as a means to differentiate pneumococci from Klebsiella pneumonia in 1884. •The Gram stain is often the first test performed in the identification of bacteria.
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- 16. Overview of the Gram stain Gram + Gram - Staphylococcus aureus