lab 4-6 Flashcards
staining
enhances contrast & resolution; reveals details
of cellular morphology & arrangement
basic stain (crystal violet)
- negative stain
- cell is stained
- positively charged attracts negative charged cells
acidic stain (nigrosin)
- simple stain (only one staining reagent)
- background is stained
- negatively charges
negative stain
- Determine morphology &
cellular arrangement (shapes and size)
– Typically for cells sensitive
to heat‐fixation during
smear preparation
– Minimal distortion of cells
(no heat‐fixation involved)
differential staining
- use more than one staining reagent to differentiate cellular structures
- allows for detection of differences between bacteria and serves as an aid in identifying bacteria.
- gram, acid-fast, spore, and capsule stain
- primary stain, counterstain, and often (though not always) a decolorizing agent.
gram stain
- most common differential stain; differentiates many bacterial types on basis of cell wall differences
- initial step in identification of unknown bacteria.
Gram positive cell
- retention of primary stain and resists decolorization
- thicker peptidoglycan and cross linkage
gram negative cell
- loss of primary stain from decolorization with alcohol
- less peptidoglycan, higher lipid content
endospores
- dormant form of a bacterium that is formed under conditions of nutrient starvation and/or other physical, metabolic stresses.
- highly resistant to heat, desiccation, and radiation
- can be visualized without staining the spore
two groups of spore-formers
1.) bacillus = soil microbes, aerobic, facultative anaerobes
2.) clostridium = anaerobic
spore stain
- uses heat to allow the dye (malachite green) to penetrate
the spore; safranin is used as a counterstain. Microscopy reveals green‐colored spores and red vegetative cells.
spore gram/simple stain
coloration of vegetative portion of the cell, while the spores appear colorless
1.) heat-fixed emulsion (up to 4)
2.) cover smear with paper and apply malachite green stain
3.) steam for 5 minutes and rinse with water
4.) counterstain with safarin for 1 minute and rinse with water
acid fast stain (kinyoun method)
- has mycolic acids (lipids in cell wall)
- carbolfuchsin solution (the primary stain), whose chemical nature allows it to penetrate the waxy acid‐fast cell wall mycolic acids; acid‐fast bacteria resist decolorization by acid‐alcohol.
- mycobacterium
non-acid fast
bacteria must be counterstained (with brilliant green) in order to be seen.
capsules
allows bacteria to avoid host defenses
capsule stain
- combination of a negative stain and simple stain; a
heat‐fixed smear IS NOT prepared.
1.) add congo red stain to slide and add organisms
2.) push the spreader slide to the other end
3.) air dry
4.) add maneval stain for 1 minute and rinse with water
selective media
contains chemical components that inhibit certain bacteria,
favors the growth of others
differential media
can visualize metabolic differences between bacterial types.
PEA
- ONLY selective to cultivate gram positive organisms
- phenylethyl alcohol reduces growth of gram negative organisms
MSA
- differential and selective
- differentiates mannitol fermenters (yellow) and non-fermenters
- high salt favors staphylococcus sp.
CNA with sheep blood
- selective for just CNA, but selective and differential with sheep blood
- promotes isolation of gram-positive bacteria by suppressing growth of gram-negative with COLISITIN and NALIDIXIC ACID
MA (macconkey agar)
- differential and selective
- inhibits gram positives (via bile salts); more gram negatives
- distinguishes between lactose (purple) & non‐lactose (colorless) fermenters.
EMB (Eosin methylene blue agar)
- differential and selective
- dyes eosin and methyl‐ene blue inhibit growth of Gram-positive organisms.
- lactose fermenters=purple
- weak lactose fermenters=pinkish-purple
- non-lactose fermenters=colorless
- strong acid= metallic green sheen (e. coli)
HEK (hektoen enteric agar)
- selective and differential
- inhibitors prevent growth of gram positive bacteria
- lactose fermentation and H2S production aids in identification of organism
- salmonella=black colonies
- shigella=translucent green colonies
- both are non-lactose fermenters - lactose fermenters (coliforms)=yellow colonies
