Study Guides (248,509)
Canada (121,598)
Biology (1,112)
BIOL 241 (60)
Final

BIOL 241L Exam notes.docx

15 Pages
672 Views
Unlock Document

Department
Biology
Course
BIOL 241
Professor
Cheryl Duxbury
Semester
Summer

Description
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
More Less

Related notes for BIOL 241

Log In


OR

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


OR

By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.


Submit