Lab 2 quiz 2 Flashcards
What are negative stains useful for?
Negative stains are useful in observing morphology, size, and arrangement of bacterial cells.
Reagents used in Negative staining?
Nigrosin
Negative Staining technique
The negative staining technique uses a dye solution in which the chromogen (nigrosin) is acidic (gives up a hydrogen ion) and carries a negative charge. Since bacteria are negatively charged as well (due to the teichoic acid on Gram positive bacteria, and the lipopolysaccharide on the Gram negative bacteria), they will repel the negatively charged chromogen. As a result, the bacterial cells will not take up the dye and will remain unstained. However, the background will be colored with the dye.
Aspetic Technique
Aseptic means free of living pathogens, ie., disease causing microbes. Aseptic technique is a way of working with microbial cultures that insures the environment, personnel, and the microbial cultures are NOT contaminated. Therefore, after you have worked with the cultures, no contamination remains and the original culture is still pure. The following is a list of general techniques and practices used when working with microbial cultures. These techniques are to be used on every occasion when working with microbial cultures. They are not listed in the order used.
Work Area Disinfection.
The work area (lab bench) is treated with disinfectant twice when working in the lab. The first disinfection is performed when first entering the lab prior to any materials being placed on the bench. The disinfection will destroy vegetative cells and viruses but generally will not destroy endospores. Therefore it is not a method of sterilization. The physical process of scrubbing the bench removes microorganisms. The work area is also disinfected after work is complete.
Bacteriological Loops and Needles.
The transfers of culture material are achieved by using inoculating loops and needles which are sterilized before and after coming in contact with the culture. The incinerator is used to sterilize the loop or needle by placing in the incinerator opening until they glow orange. The loop or needle must be cooled prior to use which is done by holding for at least 5 seconds. The loop or needle is sterilized again after transferring cultures and before returning to the bench to prevent contamination of the environment. Do not store the loop or needle in the incinerator as the loop becomes so hot the rubber holder may melt. Return the loop or needle to the bench top location.
Culture Tube Handling, Flaming, and Inoculation.
Hold culture tubes in your non-dominant hand. Prior to inserting a sterile, cooled loop or needle into the culture, the cap is removed, and the mouth is held next to the incinerator opening. Pass the culture next to the hot opening several times to produce convection currents to prevent microbes from the air from entering the culture. Hold the broth tube at an angle but still upright to minimize the chance of airborne contamination. When finished inoculating, flame the culture mouth again using the same procedure, and replace the cap on the tube. The broth tube is placed in a test tube rack and not laid on the bench top.
Petri Plate Inoculations
The plate lid is used as a shield to prevent airborne contamination by raising the cover and holding it diagonally over the plate. The loop with the inoculum is streaked over the agar surface gently to as the agar can be torn or gouged. The cover is replaced and loop is flamed.
Hand Washing (degerming).
Hands are washed as the last act before students leave the lab and after the bench top disinfection. Students also wash hands if they come into contact with bacterial cultures.
