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Final Exam Study Guide for Microbiology Lab...

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Micro lab final notes

Dilution lab -Determining Bacterial Cell Numbers 

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Background o The number of bacteria in a given sample is important when evaluating research protocols and analyses of food, water, and soil contamination. o The number of live bacteria in a sample affects the efficacy of antibiotics, antiseptics, disinfectants and shelf life. o QUALITY CONTROL Bacterial Growth Curve

o Direct Microscopic Count using a Petroff-Hauser cell counter o Relatively inexpensive o Quick o Requires high # of bacteria 106/ml o Count live and dead cells

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o Electronic Cell Counters o Fast o Easy to use o Machine is expensive o Counts ALL particles of a specific size.

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Indirect Chemical Methods o Determine the number of bacterial cells in o a population based on: o dry cell mass o the rate of oxygen consumption o the rate of consumption of a specific nutrient Estimating Bacterial numbers by use of a Spectrophotometer o Can make a graph of absorbance vs. time and match that with plate counts set up at the same time with the same culture o Indirect estimate o Requires 10-100 million bacteria to register turbidity

o Viable Plate Counts o Methods used to determine the number of live bacteria in a sample. Serial Dilution Viable Plate Count o Most frequently used method of measuring bacterial populations o Count only live cells o One bacterium grows to form one colony hence CFU (colony-forming unit) o Takes 24-48 hours

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o Procedure o After incubation we count colonies on plates containing  30-300 colonies o If more than one plate contains 30-300 colonies o determine the CFU/ml for each  then average these values  Be sure all counts to be averaged are in the same power of 10. Dilution Problems Sample o =Dilution Sample+ Diluent 3 o 1 ml=1 gram=1 cm ¿ colonies CFU o = ml plated × Dilution ml 1 −1 1 ml Mix A = =10 o 1 ml Mix A+ 9 ml H 2 O 10 1 1 ml Sample = =10−2 o 1 ml Sample+99 ml H 2 O 100 o 10−2 ×10−1 =10−3 o Serial Dilutions must be multiplied in order to determine the final dilution from the original sample. 1 =10−1=0.1 o 10

Standard Qualitative Analysis of Water 

Introduction o The human body is comprised of up to 70% water. Water is important to life!!

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o Water needs to be potable (suitable for drinking) and free of pathogens. o Chlorine and Fluorine can be added to water to disinfect it. Microorganisms in water o Water can be contaminated with bacteria, viruses, or protozoa. o Escherichia coli is used as a sentinel organism, one that alerts officials to the presence of potentially pathogenic organisms

o Presumptive Test o Determines the presence of coliforms in a water sample and determine the probable number of such organisms (the MPN- most probable number)  - Add water to lactose broths with Durham tubes. Presence of acid and gas indicates presumptive evidence for coliforms. o Suggests that water may not be suitable for drinking

o Confirmed Test o Verifies a positive presumptive test for the presence of coliforms. o - Inoculate an EMB plate from a positive lactose broth. o - Green sheen (positive) indicates that E. coli is present and water is NOT POTABLE

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o Completed Test o After a positive confirmed test, completed test is necessary to CONFIRM the presence of coliforms in the water. o Bacteria is taken from a colony on an EMB plate and is used to inoculate a lactose broth and a TSA slant. o A positive test consists of acid and gas production in the tube and presence of gram negative bacilli on the TSA slant.

o Waterborne Diseases o Bacillary dysentary shigellosis (Shigella spp.) o Typhoid fever (Salmonella typhi) o Cholera (Vibrio cholerae) o Leptospirosis (Leptospira interrogans) o Legionnaires’ disease (Legionella pneumophila) o Poliomyelitis (Picornavirus) o Hepatitis A (Picornavirus) o Naegleria meningoencephalitis (Naegleria fowleri) o Amoebic dysentary (Entamoeba histolytica) Proceedures o Instructions are on Pg. 173-176 o Water Samples are from White Stone Creek, Lake Felton, BSB Fountain, Tap Water, Pool Water, and the Baylor Marina. o Dispose of Pipette tips in the Buckets with Bleach water.

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o Food and Bacteria o Microbes are used in the production of foods like blue cheese (Penicillium) and soy sauce (Aspergillus niger) o Microbes are also responsible for food poisoning, toxicity and spoilage.

o Spoilage, Poisoning, and Toxicity o Spoilage: This is the breakdown of food by microorganisms. They can make the food look, taste, and smell different but typically wont harm you if you eat it. Ex. Pseudomonas fluorescens can survive in refrigerator temperatures and causes the slimy layer on foods such as lunch meat. o Poisoning: A bacterial infection resulting from the microorganism growing on the food, being ingested, and then multiplying in the gut. Will take up to 24-36 hours to show symptoms. Ex. Campylobacter gastroenteritis is the leading cause of food poisoning in the US. o Toxicity: Bacteria are able to grow on food and produce a toxin that, when ingested, will cause severe symptoms such as gastroenteritis and nervous system damage. Fecal coliforms o Fecal coliforms o bacteria found in fecal matter o indicate the possibile presence of pathogens o Sentinel organism: E. coli  gut of mammals  easily tested for  and its presence in foods is a rapid way to determine if fecal contamination is present. o Fecal contamination can occur in: o Crops through run off from adjacent fields, rinsing with contaminated water, and improper food handling o Animal meat through nicking the bowl at the time of slaughter or through cocontamination of ground pieces of meat. Meat should be sterile at the time of slaughter so any E. coli contamination indicates the bowel was nicked

o Poultry by run off of ice from packaging from a single contaminated bird.

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o Proceedures o Instructions are found on Pg 181-184 o When using the blender remember to hold the lid down to ensure that not splashing occurs. Prior to use make sure that is securely in place and there are no leaks. o If you have a meat sample it helps to tip the blender at 45 degrees and suck up the leading edge of the liquid to prevent fat from clogging the pipette tip. o Note: You will only be doing a 10-2, 10-3 , 10-4 Plate. o Once finished pour all blender contents back into the 180mL bottle and rinse the blender in the sinks. DO NOT pour contents down the drain!! Milk o Milk from cows is highly regulated for public safety. o Milk can only be used from a cow if there is absolutely no colostrum present. Colostrum is a high fat, high protein, high antibody substance produced for the first 3 days after a cow has given birth. o Milk can only be used for human consumption if it is in compliance with the Public Health Service FDA regulations on Grade A Pasteurized Milk

o Milk safety and Pasteurization o Grade A Raw milk can contain no more than more than 100,000 bacteria per ml prior to comingling from other milk. After comingling it can contain no more than 300,000. o Once milk has been Pasteurized it can contain no more than 20,000 bacteria per ml of milk. MILK is NOT sterile. o Pasteurization is a process of heating a liquid product briefly to reduce the number of spoilage organisms present. Originally created for preserving wine it is currently used for milk to reduce the number of spoilage organisms and eliminate

pathogens. Spoilage organisms like Streptococcus and Lactobacillus can survive pasteurization in low numbers.

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o Lactobacillus bulgarcius Types of Pastuerization o Pasteurization: Heating milk to 63˚C for 30 Minutes o Flash Pasteurization: heating milk to 72˚C for 15 seconds o Ultrapasteurization: heating milk to 127˚C for 2 seconds. Products are packaged in aseptic atmosphere in sterilized containers. It is typically done to products like Half and Half and Whipping Cream.

o Components of Milk o Lactose, a disaccharide composed of glucose and galactose. Lactaid milk contains almost no sugars, which is why it usually has a longer self-life because spoilage organism have a more difficult time growing in it o Proteins, the major source for nutrition in milk is called casein. This protein is found only in milk o Vitamin D, this has primarily been artificially added. This kind of milk is said to be “fortified” with vitamin D. o Lipids, which are used to make cellular membranes. The breakdown of lipids in milk by bacteria are what give it a rancid odor. Lipids, which are a type of fat, are blended to stay suspended in the milk. This is called homogenization. Proceedures o Instructions are on Pg 189-192 o Note: You will be doing a 10-0 ,10-1, 10-2, 10-3 Plate. This is different from the Milk lab. o Once you are finished making your plates lightly screw the cap back on the bottle and place on the autoclave cart.

ANTIBIOTIC SENSITIVITY TESTING KIRBY-BAUER METHOD 

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Automation in Clinical Microbiology o Cool Science Thing of the Week! o Laboratory automation is becoming increasingly prevalent in modern hospital and industry settings. Microbiology has always been considered a science where the ability of a human will always surpass that of a machine. o However, modern technology and merging both human users with robotics have increased production to levels that were unthinkable before without an army of microbiologists and technicians. o o Learning about these is an important step in being prepared for the future of microbiology and medicine.

o Background o The “mode of action” of a chemotherapeutic agent is how it inhibits or interferes with bacterial metabolism. o “Bacteriocidal”- an agent that KILLS bacteria o “Bacteriostatic”- an agent that INHIBITS replication (does not kill the bacteria) Therapeutic Index o Toxic dose – how much medication must be taken before undesirable side effects are seen in the patient o Therapeutic dose- how much medication must be taken before the desired effect is seen o THERAPEUTIC INDEX = toxic dose o therapeutic dose o The larger the therapeutic index, the safer the medication. I.E. Ampicillin, with therapeutic index of 100, is safer than gentamicin with therapeutic index of 3 (100 >3). o Ideally, we want to treat patients with the medication exhibiting the HIGHEST therapeutic index to minimize the risk of harmful side effects or death. o Sometimes we must choose medication with a low therapeutic index, such as with antifungal or anticancer drugs.  This is due to the fact that fungi cells are  eukaryotic and are similar to our own body;  cancer cells ARE our own cells. It is thus

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 harder to attack these unwanted cells  without harming our own. Types of Antibiotics o Sulfa drugs - sulfisoxazole, trimethoprim/sulfa o Penicillins - penicillin, amoxicillin, ampicillin o Tetracyclines - doxycycline, minocycline o Fluoroquinolones - levaquin, enrofloxacin o Macrolides - erythromycin o Aminoglycosides- streptomycin, gentamicin SULFA DRUGS o Sulfonamides - Sulfisoxazole o Bacteriostatic by COMPETITIVE INHIBITION of folic acid synthesis o ANTIMETABOLITE that competes with PABA during folic acid synthesis o Folic acid is used in the synthesis of amino acids and purines. o Broad spectrum, primarily used against Gm- organisms. PENICILLINS o Penicillins: ampicillin, amoxicillin, penicillin G o Central B-lactam ring o Inhibit transpeptidation in cross-linking of NAM units of the peptidoglycan molecule. o This weakens the cell wall resulting in cell lysis. o Most effective against Gram+ cells.

o Bacteriocidal TETRACYCLINES o Includes tetracycline, oxytetracycline, doxycycline, minocycline o Inhibits protein synthesis by binding to 30s ribosomal subunit and preventing bonding between tRNA-amino acid and mRNA codon. o Broad spectrum

o Bacteriostatic FLUOROQUINOLONES o Inhibit topoisomerase II and IV (prevent DNA replication and transcription) o Examples: Ciprofloxacin, levaquin, enrofloxacin o Broad spectrum

o Bacteriocidal

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o MACROLIDES o E.g. Erythromycin o Binds to 50s ribosomal subunit and inhibit peptide elongation o Effective against most Gram+ o and some Gramo Bacteriostatic

o AMINOGLYCOSIDES o Examples: gentamicin, streptomycin o Bind to 30s subunit and inhibit protein synthesis. o Most effective against Gram- bacteria. o Bacteriocidal

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o Kirby-Bauer Antimicrobial Sensitivity Test Procedure o Completely cover surface of Mueller-Hinton agar plate with bacteria. o Filter paper discs with antibiotic are placed on Petri dish. o Plates are inverted and incubated for 48 hours. o Record “zones of inhibition” (areas without growth)

o Zones of Inhibition o Diameter determined by:  1. Rate of diffusion of the antibiotic  2. Number of organisms on the agar.  3. Growth rate of the organism.

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 4. Degree of sensitivity of the organism to the antibiotic. Disinfectant vs Antiseptic o A chemical applied to INANIMATE surfaces to prevent contamination and infection by microorganisms o Example: Bleach and Lysol o A chemical applied to LIVING tissue to prevent contamination and infection with microogranisms o Example: alcohol and Iodine Types of Antiseptics and Disinfectants o Phenolic compounds  Ex. Phenol, Lysol, hexachlorophene o Alcohols Ex. Alcohol-based hand sanitizer o Halogens Ex. Bleach, fluoride treatments o Quaternary Ammonium Compounds  Benzalkonium chloride  Cetylpyridinium chloride o Hydrogen Peroxide Phenolic compounds o Mode of Action: Breaks down cell membranes and denatures proteins o Can be used as an antiseptic or a disinfectant depending on the chemical and concentration. Ex. Phenol is highly hepatotoxic. o Hexachlorophene is commonly used to treat acne, but is neurotoxic, esp. in infants

o Hexachlorophene Alcohols o Mode of Action: Denaturing and precipitating proteins and dissolving lipid membranes. Hydrolytic reactions. o Alcohols can be used as both disinfectants and antiseptics. Are only truly effective at 70-80%. The main component in hand sanitizers, ethyl alcohol is primarily used in the US but N-propyl is used in Europe.

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o Halogen o Mode of Action: Oxidizing agent. o Organic debris interferes with efficacy. o Halogens will attack amino acids, nucleotides, and lipids causing a disruption in function. o A small amount of iodine may not kill a cell, but mutations to DNA caused by it will eventually kill the bacteria. o Chlorine, fluorine, iodine are all used to treat drinking water. o Chlorine and bromine are used to treat swimming pool water.

Potassium Iodide main component of Tincture of Idodide, an antiseptic

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o Quaternary Ammonium Compounds o Mode of Action: Break cell membranes by lowering surface tension and denature proteins. o Quat. Compounds contain a cationic and anionic head. Typically used as an antiseptic but is ineffective against spores and viruses. Organic debris can interfere with its effectiveness

o Hydrogen Peroxide o Mode of Action: Oxidizing Agent. o Used as an antiseptic and disinfectant. While many bacteria and viruses produce catalase to defend against natural occurances of H2O2 they cannot withstand the concentrated forms we use, 3-10%. Organic debris does not interfere with its effectiveness.

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The effectiveness of antiseptics and disinfectants can be influenced by external conditions o Concentration: Alcohol is most effective at ~70% conc. o Length of Exposure: Chemicals will often take time to fully destroy bacteria and infuse into them. o Type of population targeted: Spores are resistant to many forms of disinfectants like alcohol. o Temperature: Alters the rate of chemical reactions, such as in oxidizing agents o pH: Extreme pH may alter the effectiveness of some disinfectants, like hydrogen peroxide. o Debris can also inhibit effectiveness, particularly in Quat. Ammonium compounds. The efficacy of disinfectants and antiseptics is influenced by: o Concentration o Length of exposure o Type of microbes to be destroyed o Environmental conditions, such as pH, temperature, and the presence of organic debris.

o Procedure o Obtain a plate and divide it into 5 sections o Label the plate with the appropriate organism o Swab the ENTIRE plate (“paint” the plate) with the organism. Cover every inch!! o Use CLEAN forceps to pick up an antiseptic disc and place it on the appropriate section o Flame forceps and wait to cool until picking up next antiseptic. o Repeat until all chemical discs are placed on the plate.

o Invert plate and incubate for 48 hours. o Record zones of inhibition.

What’s mostly on the test!

Micrococcus rosesus   

Gram (+) Cocci Catalase (+) Pigment of colony color – pink

Micrococcus leuteus   

Gram (+) Cocci Catalase (+) Pigment of colony color – yellow

Staphylococcus epidermidis   

Gram (+) Cocci Catalase (+) Pigment of colony color – white

Staphylococcus aureus   

Gram (+) Cocci Catalase (+) MSA (+)

Enterococcus faecalis   

Gram (+) Cocci Catalase (-) MSA (+)

Lactococcus lactis   

Gram (+) Cocci Catalase (-) MSA (-)

Streptococcus pyogenes   

Gram (+) Cocci Catalase (-) Beta hemolytic

Bacillus cereus    

Gram (+) Baillie Endospore (+) Nitrate (+) MSA plate – growth mannitol fermentation (-)

Bacillus subtilis    

Gram (+) Baillie Endospore (+) Nitrate (+) MSA plate – growth mannitol fermentation (+)

Bacillus megaterium   

Gram (+) Baillie Endospore (+) Nitrate (-)

Mycobacterium smegmatis   

Gram (+) Baillie Endospore (-) Acid fast

Corynebacterium pseudodiphthericum   

Gram (+) Baillie Endospore (-) Non-acid fast

Citrobacter freudil      

Gram (-) Fermenters H2S (+) TSI A/A Citrate (+) Indole (-) Urease (-)

Proteus vulgaris 

Gram (-) Fermenters

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H2S (+) TSI A/A Citrate (-) Indole (+) Urease (+)

Proteus mirabilis      

Gram (-) Fermenters H2S (+) TSI K/A Citrate (+) Indole (-) Urease (+)

Klebsiella pneumoniae       

Gram (-) Fermenters H2S (-) TSI A/A Non-motile Citrate (+) Indole (-) Urease (-)

Escherichia coli       

Gram (-) Fermenters H2S (-) TSI A/A Motile Citrate (-) Indole (+) Urease (-)

Enterobacter aerogenes       

Gram (-) Fermenters H2S (-) TSI A/A Motile Citrate (+) Indole (-) Urease (-)

Morganella morganil 

Gram (-) Fermenters

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H2S (-) TSI K/A Color - White Citrate (-) Indole (+) Urease (+)

Serratia marcescens       

Gram (-) Fermenters H2S (-) TSI K/A Color - Red Citrate (+) Indole (-) Urease (-)

Rhodospirillum rubrum  

Gram (-) Non-Fermenters Spirilli

Mirabella catarrhallis  

Gram (-) Non-Fermenters Cocci

Alcaligenes catcalls   

Gram (-) Non-Fermenters Bacilli Non-Saccharolytic

Pseudomonas aeruginosa    

Gram (-) Non-Fermenters Baillie Oxidative Nitrate (+)

Pseudomonas fluorescens    

Gram (-) Non-Ferm...