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Microbiology 20214

by: Jade Frederickson

Microbiology 20214 Microbiology 20214

Jade Frederickson
GPA 4.0

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Lab Exam 2 study guide
Intro to Microbiology
Dr. Curtis
Study Guide
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This 8 page Study Guide was uploaded by Jade Frederickson on Tuesday January 19, 2016. The Study Guide belongs to Microbiology 20214 at Texas Christian University taught by Dr. Curtis in Fall 2015. Since its upload, it has received 43 views. For similar materials see Intro to Microbiology in Biology at Texas Christian University.


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Date Created: 01/19/16
Lab Exam #2 Study Guide Exercise 18: Droplet Transmission  The spread of microorganisms from one individual to another via the respiratory route is  the most common means of disease transmission o The aerosol of saliva released dries down into a small particle of dust that is  inhaled and causes disease  Indicator organism: Bacteriophage T7, a virus that attacks Escherichia coli B o A solid lawn of bacteria is created by preparing the agar with a mixture of E. coli o Creates plaques, areas where the bacteria is lysed by the T7 bacteriophage o As time increases, number of plaques decrease o At 5­10 minutes, all plates have the same number of plaques because the “sneeze” has been equally distributed o The closest table will have the most number of plaques at 0­1 minutes because  they are the closest Exercise 19: Enumeration of Microorganisms in Urine  The basic assumption in determining the number of microorganisms in a sample is that  each colony represents a single cell that was initially plated  Standard practice is to use the plate with 20­200 colonies to calculate the final count  Urine plate counts used a diagnostic tool for Urinary Tract infections o Normal: 1000 or less bacteria o Infection: 100,000 or more bacteria  Infectious bacteria are usually gram­negative organisms  Dangerous because they may release lipid A upon lysing  Use a new pipet while doing the dilution so as to not introduce more  bacteria to a sample  Calculation: # colonies  *  reciprocal of dilution = number bacteria / .25 ml Exercise 20: Toilet Paper as a Barrier against Bacteria  Proper hygiene is essential for diminishing  disease among healthcare workers and  immunocompromised patients  Opportunistic pathogens can  cause disease in immunocompromised individuals  Agar surface of the plate is moist feces  Bacillus subtilis var. globigii is the digestive tract flora  Toilet paper decreases the amount of bacteria infecting your hands, but you still must  wash your hands!!! Exercise 21: Anaerobic Culture Techniques  Aerobic bacteria (aerobes) grow in the presence of free oxygen  Anaerobes cannot grow in the presence of free oxygen  Facultative organisms can grow in either condition, but prefer one or the other o  Facultative aerobe: prefers ANAEROBIC but can survive in aerobic o Facultative anaerobe: prefers AEROBIC conditions but can switch to  fermentation of anaerobic respiration if necessary  Microaerophilic organisms: require a limited amount of oxygen  Strict aerobes need free oxygen to be the terminal electron acceptor  Anaerobic Container: a GasPak foil envelope contains substances that create hydrogen  and carbon dioxide. This is placed in the container with the cultures. When the lid is  clamped down, hydrogen combines with oxygen to form water. CO2 contributes to  support the growth of fastidious anaerobes. o Methylene blue indicator strip is placed in the container to indicate the presence  of oxygen  Oxygen=blue  No oxygen=white  Thiogylcolate broth: a semi­solid medium containing sodium thioglycolate and .075%  agar  The broth removes free oxygen through reduction  Resazurin (pink) is added as an indicator  Strict aerobes grow at the top only, strict anaerobes at the bottom only and facultative  organisms grow throughout and concentrate in the area they prefer  Know which bacteria was which Exercise 22: Molecular Genetics­Transformation of Bacteria  Bacterial DNA exists in two forms, both double stranded and circular o Main bacterial “chromosome” that carries genes for necessary structures and  functions o Plasmid, a non­essential component that carries genes for antibiotic resistance  Works well for the molecular biologist, but not good for doctors because it renders antibiotics ineffective  pGLO plasmid contains the beta­lactamase gene that confers resistance to ampicillin and  allows for the bacteria to survive in the presence of ampicillin o also contains the GFP gene that is activated in the presence of arabinose— fluoresces under UV light  pM13 plasmid contains the bla gene as well, but does not contain the GFP gene  Bacterial transformation is the uptake of naked DNA by the bacteria o Most common method of retuning DNA once it has been manipulated outside the  body o Bacteria are competent if they can uptake and use the DNA in its proper function  Heat shock method: Place the tubes in a water bath for exactly 90 seconds, then rapidly  transfer them to an ice bath  The purpose of plate D is a control (for test tube 3) for both the bla gene and the GFP  gene o Nothing will grow because tube three did not get anything  The purpose of plate E is a control to show that without the bla gene and no amp, bacteria will grow, but without the pM13 gene, bacteria will not glow Exercise 23: Antibiotic Resistance  Antibiotic resistance stems primarily form the misuse of antibiotics o Misuse by patients o Overprescribing by physicians o Large dose given by hospitals o Widespread use in livestock feeds  Bacteria become resistant to antibiotics via: o Metabolic pathway o Antibiotic inactivation o Reduced permeability and active export of antibiotics o Drug target modification  Plasmids are involved in the movement of antibiotic resistance genes among bacteria  species o Requires extra energy to carry plasmids  Drug resistance also occurs as a result of natural selection o When a population is treated with an antibiotic, the sensitive bacteria are killed  and the resistant bacteria thrives as genes become operative Exercise 24: Hand­washing  Proper hygiene is essential for medical personnel who deal with immunocompromised  patients because these patients may be susceptible to opportunistic pathogens from an  individual’s intestinal flora  Four methods of hand washing o Water only o Regular soap o Antibacterial soap o Hand sanitizer  There was always a decrease in the amount of bacteria before and after wash o Water only acts as a degerming o Regular soap and antibacterial soap are a degerming and a chemical o Hand sanitizer works the best –acts as a chemical Exercise 25: Yogurt  Fermented milks are more stable under conditions of storage where refrigeration is absent or inefficient  Yogurt production utilizes the action of the members of Streptococcus thermophilus and  Lactobacillus bulgaricus o Convert milk sugar (lactose) to lactic acid plus minor amounts of other  fermentation products o Acid produced by these bacteria is sufficient enough to prevent spoilage by  bacteria that are not acid tolerant o Chilling is necessary to stop acid formation by the starter bacteria Exercise 26: Lysozyme  Lysozyme constitutes one our body’s generalized defenses against microbial infection o Present in many body secretions o Breaks down peptidoglycan, which results in the lysis of many gram positive  bacteria  Lysozyme occurs in o Tears o Saliva o Serum o Neutrophils o Macrophages o Nasal secretions o Intestinal mucous  o Urine  No direct correlation between the various lysozymes secretions  Bioassay is like a control curve used to compare unknown results to try and quantify it o Bioassay measured in lysozyme (micrograms/mL) by diameter (millimeters) o Tears have more lysozyme in them than saliva secretions o Measure zones of inhibition to determine concentration Exercise 27: Throat Cultures  Blood agar is an enriched medium composed of a generalized growth medium and 5%  sterile sheep’s blood o Usually used for throat cultures  Microbiologist usually look for species of Streptococcus, particularly the beta­hemolytics (S. pyogenes)  Breakdown of RBC results in a zone or halo around the colony  Three types of hemolysis o Alpha: greenish halo and partial breakdown of RBC o Beta: complete breakdown and a clear halo  Often correlate with sore or “strep” throat o Gamma: no breakdown of RBC  Many throat pathogens require addition CO2, thus we incubate them in a candle jar  Diluted quadrant streaking Exercise 28: Biochemical Testing  Bacteria can be classified according to immunological tests, morphological, enzyme  production and cultural tests  Tests look to see what a compounds a bacteria can utilize and what products a bacterium  will produce  Selective media: selects for the growth of specific microorganisms  Differential media: used to distinguish between different bacterial species  Starch Hydrolysis o Some microorganisms secrete exoenzymes called amylases that hydrolyze starch o After growth, flood plate with iodine, which stains starch blue o Positive Result: clear halo (yellow zone) o Negative Result: blue halo  Carbohydrate fermentation o The end products of fermentation are acid or acid and a gas o Acid production signaled by a color change from red to yellow o Used both lactose and glucose tubes  Citrate Utilization o Bacteria able to use citrate usually cause an alkaline reaction (basic—raise in pH) o Color change from green to blue because of bromophenol blue o All bacteria can metabolize citrate once internalized, but we are testing to see if  the bacterium can transport citrate  Mannitol salts o Both selective and differential o Staphylococcal species are halophiles o pH indicator added o bacteria that can metabolize the sugar mannitol will produce an acid byproduct  which will cause the phenol red in the media to turn yellow  EMB (Eosin Methylene Blue) o Both eosin and methylene blue act as inhibitors of some gram positive  microorganisms and indicators of lactose or sucrose fermentation o If able to ferment sugar, dark colonies with red centers will appear o If unable to ferment sugar, colonies remain uncolored o E. coli can be distinguished from other lactose fermenters by the presence of a  metallic green sheen (may disappear in 48 hours)  Phenylethyl alcohol o Inhibits the growth of gram negative bacteria  Urea (Urease Test) o Some bacteria possess the enzyme urease which hydrolyzes to yield ammonia and carbon dioxide o Alkaline (basic) reaction that can be detected by a pH indicator (will turn pink in  color if urea is present)  Triple Sugar Iron (TSI) o Inoculated by first stabbing the butt of the tube and then streaking the surface of  the slant o The medium tests for carbohydrate fermentation, gas production and hydrogen  sulfite production o The slant of the tube provides and array of surfaces that are either exposed to  oxygen­containing air in varying degrees (aerobic) or not exposed to air  (anaerobic) o Bacteria that ferment sugar produce acids, resulting in a yellow color change o Enterobacteria ferment sugars in the anaerobic butt of the tube o Some bacteria use thiosulfate anion as a terminal electron acceptor, reducing it to  sulfideproduces hydrogen sulfide which is visible as a black precipitate o Yellow/yellow means acid/acid reaction o Red/yellow or red/red may indicate non–lactose (sucrose) fermenters o Some bacteria use H+ as a final electron acceptor and reduce it to hydrogen gas,  forming bubble on the bottom of the slant (lifts the agar off the bottom)  Catalase test o Hydrogen peroxide is very toxic and is formed due to aerobic respiration o Catalyze breaks down H2O2 o Positive result: appearance of gas bubbles Exercise 29: Blood Typing  The combination of antibodies with its specific antigen results in readily observable  changes when carried out in vitro (outside of the body)  These tests are used to: o Demonstrate presence of an antibody in the sera of a patient o Test for compatibility of a blood transfusion, tissue grafting or autoimmune states o Identify antigens in the tissues of body fluids for blood grouping, tissue or  identification of microorganisms  Antigen­antibody reactions o Precipitation: occurs when antibodies and soluble antigens combine o Agglutination: occurs when antibody molecules combine with particulate  (insoluble) antigens (such as red blood cells)  Large clump that s are easily visible  Blood Group A o A antigens o B antibodies o Donate to A, AB o Receive from A, O  Blood Group B o B antigens o A antibodies o Donate to B, AB o Receive from B,O  Blood Group AB o A and B antigens o No antibodies o Donate to AB o Receive from A, B, AB, O  Blood Group O o No antigens o A, B antibodies o Donate to A, B, AB, O o Receive from O  Use a lancet to obtain blood sample o DO NOT share—avoid Hepatitis  The name of the antigen that gives a RBC the “positive” or the “negative” is the Rhesus  antigen o Agglutination of the Rh antigen determines “positive” or “negative” Exercise 30: Diseases  Bacillus anthracis o Gram positive rod o Causes anthrax o Spore forming o Produces three exotoxins that work together to cause disease o Inhalation, intestinal and cutaneous forms o Bioterrorism and biological warfare  Clostridium tetani o Gram positive rod o Causes tetanus o Spore formingspores enter body through a wound, germinate to vegetative cells  and produce toxins o Neurotoxin: tetanospasmin casus muscle to contract but not relax o Tetanus toxoid used in vaccination  Treponema pallidum o spirochete o Causes syphilis o Transmitted via sexual contact with an infected person, but bacteria can also enter through minor cuts or abrasions in your skin or mucous membranes o Four stages: Primary (chancre); Secondary (flu­like, rashes); Latent; Late/Tertiary (gummas, neurological)  Neisseria gonorrhoeae o Gram negative diplococci o Causes gonorrhea o Often affects the urethra, rectum or throat o Transmitted via sexual contact o Babies can be infected during childbirth when it infects the eyes  Borrelia burgdorferi o Spirochete o Causes Lyme disease and is transmitted by ticks o First appearance a bullseye’s rash o If left untreated, it can progress to chronic arthritis  Penicillum, Aspergillus, Rhizopus o Fungal respiratory illnesses o Rarely occur in those with normal immune systems o Usually seen in immunocompromised individuals such as poorly controlled  diabetes, leukemia, AIDS, etc.  Trichinella spiralis o Roundworm o Causes Trichinosis, a pork worm disease (parasite) caused by consuming raw or  undercooked meats  Trypanosoma o Flagellated protozoan o Causes African sleeping sickness o Transmitted by female tsetse flies in sub­Sahara Africa o First infects subcutaneous tissues and causes headaches, fever, joint pains and  itching o Progresses into neurological functioning when the parasite crosses over the blood  brain barrierconfusion, poor coordination, disturbance of sleep cycle o If left untreated, it can be fatal  Plasmodium o Parasite o Causes malaria, which is transmitted by the mosquito o The parasites travel through bloodstream to the liver and infect RBC  Multiply inside RBC and break open, causing high fever, shaking, chills,  flulike symptoms and anemia


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