Microbiology Lab midterm
Last Modified: 2013-02-17
One dye used to stain cells all the same color.
-can be used to tell morphology (shape) and size (although negative staining is better for size).
-cationic dyes are positively charged and are attracted by ionic forces to the negatively charged surface of bacterial cell.
-common dyes: methylene blue, crystal violet
- Gram stain & Acid fast stain
- Procedure: crystal violet, iodine, ethanol, saffranin
- gram + cells: high peptidoglycan, low lipid
- gram - cells: low peptidoglycan, high lipid
- differential stain procedure; acid fast have HIGH WAX CONTENT in their walls, which requires use of steam to allow dye to penetrate the cell.
- cells are steamed in presence of carbol fuschin, decolorized with acid alcohol, counterstained with methylene blue
- Mycobacterium & Norcardia
- (Spore staining)
- To stain the spores, the cells must be steamed to allow malachite green dye to enter the spores
- vegetative cells are easily decolorized with water, and then counterstained with safranin
- bacillus: AEROBIC, gram + rod
- clostridium: ANAEROBIC, gram + rod
- sporsarcinae: cocci
anaerobic green: endospore/ free spores of clostridium
aerobic green: endospore/free spores of bacillus
anaerobic pink: vegetative/sporangia of clostridium
aerobic pink: vegetative/sporangia of bacillus
- pour: (15-20 mL liquid agar used to pour into a plate)
- broth: (5-7 mL liquid media)
- deep: (5-7 mL media solidified upright position)
- slant (5-7 mL media solidified angled position)
- fermentation broth: broth with durham tube
- media composed of complex raw materials whose actual chemical composition is unknown
- ex. nutrient agar
media whose exact chemical composition is known and in many instances is designed for isolation, selection, of differentiation
- favors growth of one type of microoganism over another, by inhibiting the growth of one type, or by providing preferential conditions for the wanted type
- ex. PEA & DES
differentiaes or distinguishes b/w diff. types of organisms based on diff. in appearance of growth or color changes.
ex. EMB, blood agar,
- selects for the growth of gram + microorganisms
- phenylethyl alcohol inhibits gram - growth
- selects for gram - microorganisms
- Desoxycholate agar inhibits gram + growth
- differentiates for lactose fermentors
- lactose fermentors: produce acid which precipitates the bile salts in the media & absorbs the neutral red dye, appearing red.
- super fermentor of lactose (lactose +) so appears red on DES
- gram - rod
- metallic green on EMB media
- Caseinase, acid, gas (+)
- amylase, lipase, & gelatinase (-)
- selects for gram - microorganisms
- differentiates lactose +/- microorganisms
- Latose + :color change, & further differentiates b/w mixed acid fermentors, or butanediol fermentors.
EMB inhibits the growth of gram-positives and selects between lactose-fermenters and non-lactose-fermenters
- EMB agar
- produce more acid & produce colonies with dark blue centers
- E-coli produces metallic green sheen
- EMB agar
- produce less acid so that the colonies have pale pink to lavender centers
- Differentiates microorganisms based on their rxns to blood
- Gamma: (garbage) no hemolysis
- Beta: (better) complete hemolysis
- Alpha: (almost) partial hemolysis
- Hemolysis appears as clearing around the colony
- enzyme testing for: amylase (which hydrolyses starch to simple sugars)
- idicator: iodine
- appears blue/black when interacting with starch
B. Clear zone around and under bacterial growth = starch hydrolyzed by the organism’s amylase to glucose
- enzyme: caseinase (which hydrolyses casein-- a predominant protein in milk-- into amino acids)
- indicator: if it appears clear around the colonies then caseinase was present (caseinase +)
- enzyme: lipase (which hydrolyses fat into glycerol and fatty acids)
- indicator: spirit blue
- fatty acid production lowers pH to produce a dark blue precipitate when a microorganism is lipase +
- bacteria surrounded by blue: lipase +
- no blue around colony: lipase -
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