Biol 241 Lab Notes Sanya Arora BIOL 241 â Final Lab Exam Review ...
Biol 241
Lab Notes
Sanya Arora
BIOL 241 – Final Lab Exam Review Exp. 1 – Direct Microscopic Count of Microorganisms in Milk - Gram-positive, non-motile, microaerophilic/anaerobic rods and cocci - Lactobacillus, Micrococcus, Streptococcus - Stain film of milk: o Advantages: Rapid, individual bacteria + clumps counted, minimal equipment, morphology, permanent record o Disadvantages: must be high count of bacteria in milk, dead cells/debris are counted as well (difficult to discern), examinations are tiring - Procedure: Vortex (breaks up clumps) → slidebrite (remove fat globules), stain → microscope factor calibration o Microscope factor: relationship of the field to a milliliter o Get diameter → get area → convert to cm2 → 1 cm2 divide by area of fields of view = # of fields of view in 1 mL → divide by volume of milk spread = fields/mL = microscope factor Exp. 2 – Standard Plate Count of Milk - Used to estimate bacterial populations; official method for determining the sanitary quality of milk - Serial dilutions and then spread-plate/pour-plate - Individual cell = colony = viable microorganisms - Advantages: only viable organisms, accurate for low bacterial content - Disadvantages: only microorganisms capable of growth in that media counted, single cells/clumps of cell may have become colonies - Pasteurisation = not sterilised - Dilution factor x microorganisms = CFU/mL Exp. 3 – Microbiological Analysis of Cheese - LAB (Streptococcus/Lactobacillus) used as starter colonies o Produce organic acids causing souring/flavours - Other colonies added; causes curds (protein) to form due to enzymatic activity - Liquid whey drawn off leaving curd - Three types: o Soft-acid: cottage/cream o Hard-rennet: Swiss/cheddar o Semisoft-rennet: Camembert - Defects: o Gas formation from Clostridium, E. coli, Enterobacter o Spoilage due to dairy mold Geotrichum, Cladosporium, Penicillium - APT agar: all-purpose tryptose, all MO grow - EMB: selects for coliforms - Malt extract: selects for moulds Exp. 4 – Food Illnesses Caused By Staphylococci and Salmonellae - S. aureus: heat stable enterotoxin (exotoxin) → releases it outside of the MO so large amounts necessary for sickness - Salmonella: endotoxin → on the LPS membrane as lipid A so only small amounts needed - Week 1: isolate using Staph 110 plates and selenite-cysteine broth - Week 2: o Count plates, get yellow golden colony, transfer to brain heart infusion (BHI) broth o Streak plate SC culture to the MacConkey agar Both selenite-cysteine and tetrathionate Brilliant Green broths used, Brilliant Green Sulfa and MacConkey agars used → enrichment steps because usually only small amounts are found in a food sample - Week 3:
[Type text]
[Type text] o o
[Type text]
Coagulase test for Staph; most exotoxin strains will have coagulase and will form a clot Clot formation allows Staph to trap itself inside animals No lactose fermentation, transparent-pink in colour, transfer to TSI slant No lactose/sucrose, uses dextrose, produces H2S and gas in TSI slant
Exp. 5 – Determination of Coliforms in Water by the Most Probable Number (MPN) Test - Sanitary quality = types of MOs present, not number; water is safe to drink even with many MOs o Potable: no human pathogens present - Many pathogens = fecal origin, introduced through sewage; ∴ detection using organisms that live in GI tract and rarely anywhere else : coliforms o Easily detected, large numbers, non-pathogenic, presence indicates presence of fecal matter, not of pathogens 1. Presumptive test: o Inoculate water into lauryl tryptose; if fermented + gas = unsafe o Clostridium/Bacillus may cause gas formation as well 2. Confirmed test: o Tubes with gas selected and inoculated into brilliant green lactose bile If gas = positive; much more selective for coliforms o Streak on to EMB agar or Endo; look for metallic green/pink colonies with dark centres E. coli better b/c Enterobacter lives in grains/soil; EMB inhibits Gram-positives 3. Completed test: o Inoculate into lauryl tryptose + Gram-stain = non-spore forming, bacilli, Gram-negative = coliforms - Use MPN (number of positive tubes w/ positive and different dilutions = gives a number) Exp. 6 – Determination of Coliform Numbers in Water by the Membrane Filter Technique - Endo medium: selective for coliforms; extraneous growth would cause the coliform colonies to be hidden - Coliforms = lactose fermenters → aldehydes o Medium contains basic fuchsin, reacts with aldehydes = shiny green - Two types of coliforms: o Total: green sheen + ferment + acid + gas o Fecal: ferment + acid + gas + blue colonies using m-FC broth on membrane filters; higher temperature than total coliforms (heat selection) More indicative of fecal pollution - Advantages compared to tubes: greater sensitivity b/c larger volumes tested, short time, higher reproducibility, no confirmed/completed tests needed - Disadvantages: water must be free from extraneous matter such as cyanobacteria b/c the filter will be plugged quickly Exp. 7 – Comparison of Colony Appearance of Coliforms and Other Bacteria Grown on EMB and Endo Agars - EMB and Endo contain dyes that inhibit growth of Gram-positives - E. coli/C. freundii: dark green metallic sheen on both = coliform lactose fermentation - Salmonella typhimurium/P. aeruginosa: dark, white/pink colonies = non-lactose fermenters o P. aeruginosa nutrient agar = fluorescent green - B. subtilis/S. aureus: no growth = Gram-positive o S. aureus nutrient agar = yellow golden colonies Experiment 8 – The IMViC Test - Separates coliforms; especially E. coli and Enterobacter - Indole, methyl red, Voges-Proskauer (acetylmethylcarbinol production), citrate (carbon source) o E. coli: ++--
Biol 241
-
-
Lab Notes
Sanya Arora
o E. aerogenes: --++ o C. freundii: -+-+ MR-VP medium, tryptone and Simmon’s citric agar slant Indole: tryptophan degradation → indole, pyruvic acid, ammonia → indole reacts with Kovac’s reagent = red Methyl-red: tests for mixed acid vs. 2,3-butanediol fermentation of glucose o E. coli: mixed acid = more acidic = indicator is red when pH
Lab Notes
Sanya Arora
BIOL 241 – Final Lab Exam Review Exp. 1 – Direct Microscopic Count of Microorganisms in Milk - Gram-positive, non-motile, microaerophilic/anaerobic rods and cocci - Lactobacillus, Micrococcus, Streptococcus - Stain film of milk: o Advantages: Rapid, individual bacteria + clumps counted, minimal equipment, morphology, permanent record o Disadvantages: must be high count of bacteria in milk, dead cells/debris are counted as well (difficult to discern), examinations are tiring - Procedure: Vortex (breaks up clumps) → slidebrite (remove fat globules), stain → microscope factor calibration o Microscope factor: relationship of the field to a milliliter o Get diameter → get area → convert to cm2 → 1 cm2 divide by area of fields of view = # of fields of view in 1 mL → divide by volume of milk spread = fields/mL = microscope factor Exp. 2 – Standard Plate Count of Milk - Used to estimate bacterial populations; official method for determining the sanitary quality of milk - Serial dilutions and then spread-plate/pour-plate - Individual cell = colony = viable microorganisms - Advantages: only viable organisms, accurate for low bacterial content - Disadvantages: only microorganisms capable of growth in that media counted, single cells/clumps of cell may have become colonies - Pasteurisation = not sterilised - Dilution factor x microorganisms = CFU/mL Exp. 3 – Microbiological Analysis of Cheese - LAB (Streptococcus/Lactobacillus) used as starter colonies o Produce organic acids causing souring/flavours - Other colonies added; causes curds (protein) to form due to enzymatic activity - Liquid whey drawn off leaving curd - Three types: o Soft-acid: cottage/cream o Hard-rennet: Swiss/cheddar o Semisoft-rennet: Camembert - Defects: o Gas formation from Clostridium, E. coli, Enterobacter o Spoilage due to dairy mold Geotrichum, Cladosporium, Penicillium - APT agar: all-purpose tryptose, all MO grow - EMB: selects for coliforms - Malt extract: selects for moulds Exp. 4 – Food Illnesses Caused By Staphylococci and Salmonellae - S. aureus: heat stable enterotoxin (exotoxin) → releases it outside of the MO so large amounts necessary for sickness - Salmonella: endotoxin → on the LPS membrane as lipid A so only small amounts needed - Week 1: isolate using Staph 110 plates and selenite-cysteine broth - Week 2: o Count plates, get yellow golden colony, transfer to brain heart infusion (BHI) broth o Streak plate SC culture to the MacConkey agar Both selenite-cysteine and tetrathionate Brilliant Green broths used, Brilliant Green Sulfa and MacConkey agars used → enrichment steps because usually only small amounts are found in a food sample - Week 3:
[Type text]
[Type text] o o
[Type text]
Coagulase test for Staph; most exotoxin strains will have coagulase and will form a clot Clot formation allows Staph to trap itself inside animals No lactose fermentation, transparent-pink in colour, transfer to TSI slant No lactose/sucrose, uses dextrose, produces H2S and gas in TSI slant
Exp. 5 – Determination of Coliforms in Water by the Most Probable Number (MPN) Test - Sanitary quality = types of MOs present, not number; water is safe to drink even with many MOs o Potable: no human pathogens present - Many pathogens = fecal origin, introduced through sewage; ∴ detection using organisms that live in GI tract and rarely anywhere else : coliforms o Easily detected, large numbers, non-pathogenic, presence indicates presence of fecal matter, not of pathogens 1. Presumptive test: o Inoculate water into lauryl tryptose; if fermented + gas = unsafe o Clostridium/Bacillus may cause gas formation as well 2. Confirmed test: o Tubes with gas selected and inoculated into brilliant green lactose bile If gas = positive; much more selective for coliforms o Streak on to EMB agar or Endo; look for metallic green/pink colonies with dark centres E. coli better b/c Enterobacter lives in grains/soil; EMB inhibits Gram-positives 3. Completed test: o Inoculate into lauryl tryptose + Gram-stain = non-spore forming, bacilli, Gram-negative = coliforms - Use MPN (number of positive tubes w/ positive and different dilutions = gives a number) Exp. 6 – Determination of Coliform Numbers in Water by the Membrane Filter Technique - Endo medium: selective for coliforms; extraneous growth would cause the coliform colonies to be hidden - Coliforms = lactose fermenters → aldehydes o Medium contains basic fuchsin, reacts with aldehydes = shiny green - Two types of coliforms: o Total: green sheen + ferment + acid + gas o Fecal: ferment + acid + gas + blue colonies using m-FC broth on membrane filters; higher temperature than total coliforms (heat selection) More indicative of fecal pollution - Advantages compared to tubes: greater sensitivity b/c larger volumes tested, short time, higher reproducibility, no confirmed/completed tests needed - Disadvantages: water must be free from extraneous matter such as cyanobacteria b/c the filter will be plugged quickly Exp. 7 – Comparison of Colony Appearance of Coliforms and Other Bacteria Grown on EMB and Endo Agars - EMB and Endo contain dyes that inhibit growth of Gram-positives - E. coli/C. freundii: dark green metallic sheen on both = coliform lactose fermentation - Salmonella typhimurium/P. aeruginosa: dark, white/pink colonies = non-lactose fermenters o P. aeruginosa nutrient agar = fluorescent green - B. subtilis/S. aureus: no growth = Gram-positive o S. aureus nutrient agar = yellow golden colonies Experiment 8 – The IMViC Test - Separates coliforms; especially E. coli and Enterobacter - Indole, methyl red, Voges-Proskauer (acetylmethylcarbinol production), citrate (carbon source) o E. coli: ++--
Biol 241
-
-
Lab Notes
Sanya Arora
o E. aerogenes: --++ o C. freundii: -+-+ MR-VP medium, tryptone and Simmon’s citric agar slant Indole: tryptophan degradation → indole, pyruvic acid, ammonia → indole reacts with Kovac’s reagent = red Methyl-red: tests for mixed acid vs. 2,3-butanediol fermentation of glucose o E. coli: mixed acid = more acidic = indicator is red when pH
