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BIOL 241L Exam notes.docx

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BIOL 241
Cheryl Duxbury

BIOL 241L – Introduction to the Microbial World Experiment 1 – Direct Microscopic Count of Microorganisms in Milk - Microbial population of milk typically composed of Gram-positive, non-motile, microaerophilic or anaerobic rods or cocci o Ex. Lactobacillus, Micrococcus, and Streptococcus - Microbial content of milk determined by microscopic examination of stained film of milk sample - Advantages: o Results are available rapidly o Individual bacteria as well as clumps can be counted o Minimum amount of equipment required o Morphological types of bacteria can be determined o Slides can be kept as a permanent record - Disadvantages: o Counts cannot be made accurately on low-count milk o Dead cells present in pasteurized milk are counted and provide misleading results o Continuous microscopic examinations are tiring o Cells may be difficult to discern and extraneous debris may be confused with bacterial cells - Use 0.005 mL of appropriate milk sample into three-1 cm areas on the slide - Sterilized inoculating loop to spread drop evenly over the area and air dry over beaker of boiling water - Cover slide with SlideBrite to remove fat globules - Rinse with 95% ethanol to remove SlideBrite then stain with methylene blue and decolourize with 95% ethanol - Calibration of Microscope – Microscope Factor (MF) o Used to know what part of a mL of milk is represented in that field o Microscope Factor: relationship of the field to a milliliter - SAMPLE CALCULATION 2 2 o Area = πr (in cm ) o Number of fields in 1 cm = 1.0 cm ÷ area per field o Microscope Factor = number of fields in 1 mL (in fields/mL) = number of fields in 1 cm area ÷ vol. of milk over 1cm - Cells per mL of milk = Average # of cells/field x MF Experiment 2 – Standard Plate Count of Milk - Standard Plate Count: agar-plate method for estimating bacterial populations - Used as official method for determining sanitary quality of milk - Serial dilutions prepared and spread over surface of agar growth medium or mixed with molten- agar growth medium - Microorganisms present in sample form colonies on and/or in the agar media - Total number of colonies formed is equivalent to number of viable microorganisms in diluted sample - Serial dilutions: to ensure that a sample will be obtained and produce separate and distinct colonies for counting - Advantages: o Counts only viable organisms – accurate for use with milk having a low bacterial content - Disadvantages: o Only detects organisms capable of utilizing particular nutrients supplied and growing under set incubation conditions o Colonies may be derived from either single cells or clumps of cells - SPC should only be an estimate of total population present in sample - Plates incubated at 37 C for 48 hours - Number of organisms per mL = number of colonies x dilution factor (inverse of dilution) - Use Quebec colony counter to count each plate with 25-250 colonies - Dilution: how much you’ve diluted the sample - Dilution factor: ratio of final volume over original (1/100  dilution/dilution factor) - SPC should have lower counts than DC Experiment 3 – Microbiological Analysis of Cheese - Lactic acid-producing bacteria initiate cheese-making process o Ex. Streptococcus and/or Lactobacillus - Organic acids produced are responsible for acidification (souring) of milk - Microorganisms added to sour milk and enzymes produced will cause protein molecules (curd) to separate from mixture - Whey (liquid) drawn off to leave the curd for processing - Soft: acid-curd cheese – cottage, cream cheese - Hard: rennet-curd cheese – Swiss, cheddar cheese - Semisoft: rennet-curd cheese – Camembert - Cheddar cheese defect – formation of gas, moulds o Gas = coliforms o Moulds = Penicillium - DEFECT: formation of gas due to presence of gas producing organisms o Ex. Clostridium and/or coliform bacteria (ex. Escherichia coli, and Enterobacter aerogenes) - Spoilage may also be due to “dairy mould” o Ex. Geotrichum, Cladosporium and Penicillium - Investigate presence of coliform bacteria and/or moulds which can cause spoilage - Use APT agar, EMB agar, Malt Extract agar o o - APT – 30 C; EMB – 37 C; MAE – room temperature - APT (all purpose tween) = non selective media - EMB (eosin methylene blue agar) = selective for gram negative, differentiates between E. Coli and Enterobacter - MEA (malt extract agar) = selective for fungi - RESULTS o APT: raised, dry – long rods o EMB: raised, dry – round o MEA: raised, fibrous, wrinkled – clumped round bacteria Experiment 4 – Food Illnesses Caused by Staphylococci and Salmonellae Unknown #2 - Food poisoning caused by enterotoxin-producing strains of Staphylococcus aureus o May enter food from sources such as food-handlers, excrete heat stable exotoxin into food product o Causes food poisoning or intoxication - GOAL: distinguish between 2 MO’s responsible for food-related illnesses o Staphylococcus  exotoxin o Salmonella  endotoxin - Ingestion of pathogenic microorganisms may cause them to grow in the intestinal tract and produce toxic substances o Ex. Salmonella – liberate endotoxins - Either Staphylococcus aureus or Salmonella sp. Added to simulate contaminated food in this experiment - Week 1: Isolation o Selenite-cystine broth – used to enrich Staphylococcus aureus  Used to isolate Salmonella o White colonies = Staphylococcus epidemidis o Yellow/orange colonies – Staphlyococcus aureus (THIS IS WHAT WE WANT) o Staphylococcus 110 plates have higher salt concentration to encourage growth - Week 2: Enumeration and Enrichment o Colony on Staph 110 plate chosen and transferred to brain heart infusion broth (BHI) – type of enrichment - Detection of Salmonella o In actual practice, Selenite-Cystine and Tetrathionate Brilliant Green enrichment broths and Brilliant Green Sulfa and MacConkey agars are used o Streak loopful of Selenite-Cystine culture on plate of MacConkey agar - Week 3: Identification of Staphlyococcus by Coagulase o Perform coagulase test by adding 2 drops of culture into sterile test tube o POSITIVE: heavy, firm clot – means there were organisms present in the sample o NEGATIVE: no clot – no organisms present - Further Detection of Salmonella o Salmonella sp. do not ferment lactose – will be transparent to pink in colour o Stab inoculate and streak slant - Week 4: o Salmonella reactions for Triple Sugar Iron Agar (TSI) found in table below o Salmonella isolates can be further screened using additional biochemical and serological tests Components of TSI Observation Reaction shown by majority of Salmonella Lactose and/or Sucrose Positive: yellow slant Negative utilization Negative: colour of slant becomes more intensely red Dextrose utilization Positive: yellow butt with or without Positive gas formation (if black, dextrose must have been positive) Negative: colour of butt remains unchanged H 2 production Positive: blackening of butt often Positive extending into slant Negative Reaction: no blackening Gas formation Positive: formation of gas pockets in Positive medium Negative: no gas pockets in medium Experiment 5 – Determination of Coliforms in Water by the Most-Probable-Number Method (MPN Test) - Natural waters usually contain large numbers and large varieties of microorganisms - Sanitary quality determined primarily by kinds of microorganisms present (not determined by number) - Public Health Service Drinking Water Standards state: drinking water should be entirely free from pathogenic microorganisms - Many pathogens are of fecal origin and introduced into potable water via sewage pollution - Coliform bacteria are used as indicator organisms because sewage always contains them in large numbers o Also detected more easily than other pathogens - Most coliforms are non-pathogenic, and detection in drinking water indicates presence of fecal matter and not necessarily harmful microorganisms - Tests are conducted in sequence o Presumptive test: consists of inoculating a known amount of water sample into multiple lauryl typtose broth tubes  If fermented with production of gas within 24-48h, water is assumed to be contaminated with fecal materials – therefore unsafe to drink  Occasionally, positive result from presumptive test may caused by Clostridium or Bacillus (not coliforms) o Confirmed test: all tubes of original sample with gas formation are selected  Add a loopful from each tube into tube of brilliant green lactose bile broth (more selective for coliform detection)  If shows production of gas, confirmed test is considered positive  ALTERNATIVE: streak culture from positive and doubtful tubes onto Eosin Methylene Blue (EMB) or Endo agar  EMB’s methylene blue inhibits gram-positive bacteria  Gram-negative lactose fermenters = coliforms will grow on the medium and produce nucleated colonies  If typical coliform colonies, test indicates gas in the presumptive tubes was most likely produced by coliforms  E. coli not normally present in soil and is more reliable o Completed test: usually selects a well-isolated, typical coliform colony  Inoculates a portion of the colony into lauryl tyrptose broth  Gram stain  Gram negative = nonsporeforming bacilli  Positive = produced acid and gas from lactose - RESULTS: o Presumptive Test: Durham tube needs > 10% gas  Results determined by number of positive tests  MPN = most probable number of coliforms present in sample o Confirmed Test: observe presence of at least 10% gas  Use MPN Index to get “confirmed” coliforms/mL  Alternative Confirmed: if no coliform colonies present, water considered safe to drink  Greenish metallic sheen (E. coli)  Pink colonies with dark centre (E. aerogenes) o Completed Test: if gas appears in lactose-broth tube and microscopic examination reveals gram-negative nonsporeforming bacilli, presence of coliform Experiment 6 – Determination of Coliform Numbers in Water by the Membrane Filter Technique - PURPOSE: isolate and grow coliforms from a selected water source by trapping them on surface of membrane filter for culturing into identifiable colonies - Endo medium – selective in discouraging growth of other species of bacteria o “Raw” water sources contain many different species of microorganisms - Coliform bacteria have ability to break down lactose – forms aldehydes - Endo medium has lactose and other nutrients as well as basic fuchsin o Basic Fuchsin: stain to react with aldehyde molecules to produce a complex with shiny green coating o Green metallic sheen = coliform bacteria - Coliforms divided into 2 types o Total coliforms: organisms that ferment lactose with production of acid and gas – forms green sheen on membrane filters using Endo broth o Fecal coliforms: able to grown and ferment lactose with production of gas and acid – forms blue colonies on membrane filters using mFC broth  Counts considered as more indicative of fecal pollution by man and other warm- blooded animals - Advantages: o Greater sensitivity because larger volumes of water can be tested o High degree of reproducibility of results o Shorter time for obtaining them because doesn’t require confirmatory or completed tests - Disadvantage o Water should be free from extraneous matter o Filter may be clogged easily - ENDO BROTH = GREEN METALLIC SHEEN - m-FC BROTH = BLUE COLONIES (see fecal coliforms as dark blue because of aniline blue dye) Experiment 7 – Comparison of Colony Appearance of Coliforms and Other Bacteria Grown on EMB and Endo Agars - EMB and Endo do not only have Escherichia coli growing - Other bacteria may grown readily on medium but produce colonies differing in appearance from E. coli – growth of other organisms may be inhibited - Divide plate into 3 sections and streak with MO - Nutrient Agar: serves as control - EMB: contains eosin methylene blue and lactose - Endo: contains fuschin dye and sodium sulphite o Red NOT due to acid but acetaldehyde (Fixed by sodium sulphite and in presence of fuschin forms dark red colour) - RESULTS o Nutrient Agar – all organisms had exceptional growth o EMB Agar  Escherichia coli, Pseudomonas aeruginosa (+++)  Salmonella typhimurium, Citrobacter freundii (++)  Bacillus subtilis, Staphylococcus aureus (+) o Endo Agar  Escherichia coli (+++)  Salmonella typhimurium, Pseudomonas aeruginosa (++)  Citrobacter freundii, Bacillus subtilis, Staphylococcus aureus (-) Experiment 8 – The IMViC Test - Developed as means of separating various types of coliform organisms (Escherichia coli and Enterobacter aerogenes) - I – indole production o Differentiates organisms that can separate indole ring from amino acid tryptophan from those that cannot o Pyruvic acid and ammonia are utilized by bacteria and indole ring is not so it will be accumulated in the medium o Positive test: tryptophan  3H + pyruvic acid + indole ring o Indole ring + Kovac’s reagent  RED - M – Methyl red test o E. coli ferments glucose to acidic products – imposes a self-limiting condition (pH below 4.5) o Other bacteria produce products like butylene glycol and will exhaust the glucose supply and attack peptone liberating ammonia (produce basic environment) o Positive test: glucose  pyruvic acid  acidic products (acetic, succinic, formic) o Negative test: glucose  pyruvic acid  neutral end products (butylene) o Methyl red indicator: over 4.5 pH = yellow = negative Under 4.5 pH = red = positive - Vi – Voges-Proskauer reaction (production of acetylmethylcarbinol) o Designed to detect acetylmethylcarbinol (acetoin) from sugar fermentation o Positive test: glucose  pyruvic acid  acetoin  butanedol  Acetoin + alpha naphtol + KOH = RED o Cherry red = positive o Brown = negative - C – ability of organism to use citrate as only carbon source o Medium has only one carbon source (sodium citrate) o Bacteria utilize sodium citrate and bring pH to basic side o Bromothymol blue indicator from green to blue o Blue = alkaline o Green = neutral - RESULTS o Indole production  Escherichia coli positive  Enterbacter aerogenes, Citrobacter freundii negative o Methyl red test  Escherichia coli positive  Enterbacter aerogenes, Citrobacter freundii negative o Voges-Proskauer reaction  Escherichia coli negative  Enterbacter aerogenes, Citrobacter freundii positive o Citrate Test  Escherichia coli, Citrobacter freundii negative  Enterbacter aerogenes positive Experiment 9 – Determination of Chlorine Demand - Physical/chemical agents may be used to purify waters o Chlorine added to disinfect - Solutions containing chlorine (like sodium hypochlorite – Javex) depend on liberation of free chlorine and subsequent oxidation of enzymes and proteins by oxygen for their effectiveness - Hypochlorite  hypochlorous acid  hydrochloric acid + nascent oxygen (oxidizing agent) - Chlorine is a strong oxidizing agent – affinity for organic compounds - Water supplies with organic matter have a “chlorine demand” – bactericidal in nature o Chlorine demand: amount of chlorine used up by chemical
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