BIO 211L - Quiz 5 PDF

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Professor TA: Briana Tatum...

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Quiz 5 Review Sheet - Exercises: 18,19,20,27,29, The Vaccine War Exercise 18 - Gram-positive Cocci (Staphylococci) Staphylococci Genus Characteristics - Staphylococcus defined: “coccus” = spherical and “staphylo” = arranged in clusters/grapes - Gram Positive - Catalase – positive (contains an enzyme called Catalase that breaks down the hydrogen peroxide) - Colonies: opaque, ranging from white to cream colored to yellowish 2H2O2

catalase

H2O + O2

Pathogenesis - S. epidermidis and S. aureus are part of the normal human flora - Staphylococcus can also live on inanimate objects in many environments (“fomites”) - Normally do not cause disease, but they are opportunistic pathogens - Can be acquired nosocomially o Contracted from microbes in hospital environment (on formites) o Contracted from health care workers (“carriers”) from their normal flora Staphylococcus Aureus - Resident of skin and nasal cavity - Pathogenic due to presence of certain virulence factors - Coagulase: combines with prothrombin to form staphylothrombin, which then causes blood plasma to clot (fibrinogen to fibrin) - Hemolysin/staphylococcus alpha toxin: toxin that causes the complete lysis of red blood cells Staphylococcus Medium - Selective NaCl medium - Contains sodium chloride (salt), which restricts the growth of many different types of bacteria (halophiles) - Staphylococcus can withstand salt concentrations so the medium will selectively flow them Main experiments used for Staph species Mannitol Salt Agar (MSA) - Selective medium for GM+ staph; high salt concentrations inhibit GM – bacteria - Medium contains a color pH indicator that turns from red to yellow - If bacteria can ferment mannitol, acid are produced - Typical hallmark of pathogenic Staph - If acids are produced, pH turns acidic (lower pH) - When pH changes to acidic, we see a color change Expected Results MSA - S. epidermidis, non-pathogenic, cannot ferment mannitol, medium stays red

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S. aureus, ferment mannitol, medium becomes yellow

Staphylococcus Medium 110 (SM110) – high salt conc. + pigment production - Selective medium for GM + staph; high salt concentration - Differential medium : differentiates species of staphylococcus on the basis of pigment production (ie . different species will produce different pigments) Expected Results SM110 - S. epidermidis : no pigment production - S. aureus : yellow pigment

Coagulase test Control S. aureus S. epidermidis

DNA Agar plate (DNAse)

DNASE NEGATIVE = BLUE DNASE POSITIVE = PINK Hemolysis - Staph on BAP (blood agar plates)

BETA = Complete hemolysis GAMMA = No hemolysis Exercise 19 - Streptococci Streptococci Genus Characteristics - “Strepto” = strip/string/chain - Catalase negative - Gram positive cocci in chains - Grow well in low O2 and high CO2 environments (anaerobes) - Some part of normal flora and some pathogenic Streptococci and Humans - Normal flora in mouth: Corynebacterium, Hemophilus, Nisseria, Staphylococcus, and Streptococcus - Streptococcus salivarius: Causes dental plaque and cavities - Streptococcus mutans: Causes dental caries (tooth decay) Streptococci and Humans Streptococcus pyogenes - Strep throat - Necrotizing fasciitis (flesh eating bacteria) - Scarlet fever - Rheumatic fever Enterococcus spp. - Look similar to streptococcus features - 10% of all nosocomial infections - Vancomycin resistant enterococci (VRE) Differentiation of streptococcus Spp. 1. Based on their hemolytic properties (rupture or destruction of red blood cells) - uses BAP and candle jar (know why a candle jar is important! High CO2/Low O2 environments?) 2. Lancefield serotyping (based on specific carbohydrates in the bacterial cell wall detected with antibodies) Hemolysis α hemolysis: Partial hemolysis - S. salivarius, S. mutans

β hemolysis: Complete hemolysis (most pathogenic) - S. pyogenes, S. equi γ hemolysis: No hemolysis - S. salivarius, S. mutans α- and γ-hemolytic = normal flora β-hemolytic = mostly pathogenic Carriers - Carriers are people that can harbor a pathogen without exhibiting signs of illness and transmit them to others - A percentage of people are asymptomatic carriers of pathogenic Streptococcus - Example: 20-25% of pregnant women carry it in their vagina or rectum (usually tested BEFORE giving birth for Group B Streptococcus (GBS) natural or cesarean birth) Isolation of Streptococcus Spp. - Use blood agar plate and candle jar - Streptococci grow very well (are enriched) in the candle jar, which has very high CO2 concentrations are low O2 concentrations due to a burning candle but not completely anaerobic Comparing STAPHYLOCOCCUS Staphylococcus GM + cocci Arranged in clusters Catalase + Some normal flora (e.g. Staphylococcus epidermidis) Some pathogenic (e.g. Staphylococcus aureus)

Streptoccus GM + cocci Arranged in chains Catalase – Some normal flora (e.g. Streptococcus salivarius) Some pathogenic (e.g. Streptococcus pyogenes)

Main experiments used to differentiate Strepto species Catalase plasma test

Hemolysis on BAPs

E. FAECALIS ON BAP (SLIGHT ALPHA TO GAMMA)

S. VIRIDANS ON BAP (ALPHA HEMOLYTIC)

S. PNEUMONIAE ON BAP (ALPHA HEMOLYTIC)

S. EQUI ON BAP (BETA HEMOLYTIC)

S. SALIVARIUS ON BAP (SLIGHT ALPHA TO GAMMA) P discs

S. PNEUMONIAE (SENSITIVE TO P DISC)

E. FAECALIS (RESISTANT TO P DISC)

S. EQUI (RESISTANT TO A DISC AND SENSITIVE TO SXT DISC) 6.5% NaCl Broth

Positive

Negative

Bile Esculin Test

Negative

Positive

Exercise 20 - Urine Culture Sterile and Non-sterile portions of the Urinary Tract

Urinary Tract susceptible to Urinary Tract Infection (UTI) - Escherichia coli -

Proteus spp.

-

Pseudomonas spp.

-

Enterobacterium spp.

-

Streptococcus spp.

-

Staphylococcus spp.

Universal Precautions (with bodily fluid) *PPE = Personal Protective Equipment 1. Wash your hands before and after handling 2. Disinfect before and after working 3. Wear gloves (latex or vinyl) 4. Wear a lab coat and closed toed shoes 5. Use a protective barrier (Plexiglas shield) 6. Dispose of samples properly Urinary Tract Infection Tests 1. Urine Dipstick Test: Uses a strip of paper (akin to pH strips) with different chemical sensors; interpretation based on colors, refer to chart - Easy and quick test for UTI and other health conditions (time sensitive) 2. Dilution Plating: Plate a urine sample onto plates; count colonies to interpret the results - Collected urine is plated on TSA plate to isolate and enumerate the number of bacteria in the urine specimen - Use plastic loops (don’t flame it) to collect urine and streak urine across plate Calculate Bacterial Concentrations (serial dilutions) - Normal (healthy person) = Number < 10,000 CFU/mL of varieties of bacteria - Possible UTI = Number > 10,000 CFU/mL of different varieties of bacteria; plate onto differential and selective media for identification

Exercise 27 – Microbial Spoilage of Meat Food Spoilage Food Spoilage microbes adapt to appropriate environmental conditions and are equipped to utilize certain types of food - Possess enzymes capable of degrading human foodstuffs into basic nutrients they need to survive Meat – high in nutrients (fats, proteins, minerals, ect…); ideal for bacterial growth (if they have the enzymes to break down and use these nutrients) Meat Contamination Sources: - Slaughter (environmental bacteria, normal animal microbial flora from other body parts) - Processing and packing (food processing surfaces, machinery, human contact, contaminated water) - Food preparation surfaces (countertops, cutting boards) Types of Contamination - Molds - Yeasts - Bacteria (Pseudomonas, Serratia, Vibrio, Bacillus, Clostridium, Micrococcus, Enterobacter, Klebsiella, Lactobacillus, Staphylococcus) - Grows at low temperatures to survive refrigeration process Food Spoilage Bacteria - Low Temperature Mesophiles (grow around 20-25°C, but also survives at 37°C) - Psychrophiles (grow -5 to 20°C; not able to grow at room temperature well, but at low temps can degrade food products) - Psychrotrophs (facultative psychrophiles; can grow above 20°C but can grow at low temperatures)

Exercise 29 - Milk Microbiology Pasteurization - Method for preserving milk and other dairy products - Process: heating milk to a temperature below the boiling point (usually 60 degree C) for 20 minutes; brought to 10 degree C

- Flash pasteurization: 72°C for 15 seconds; rapid cooling to 10°C - Pasteurization does not kill all microbes – but it reduces their numbers Standard Plate Counting - Method used to determine the quality of milk - High counts may include: sick cows, unsanitary handling, inadequate storage o TGEA plates used to determine bacterial concentration of milks o Determines quality of milk o High quality = pasteurized milk o Low quality = raw milk TGEA: typtone glucose extract agar - Make dilution first! Keep TGEA in water bath until you are ready to use - Milk milk or diluted milk with liquid TGEA, then pour into a sterile Petri plate - Let agar solidify, then we will incubate at 35C

Reduction of Milk - why do low quality milks have a higher reduction? (High concentration of bacteria) - Actively growing bacteria consume oxygen in milk, which reduces the milk (chemical reduction: gain of electrons/loss of oxygen (the opposite of oxidation)) - Measuring reduction is a measure of the number of bacteria in a milk sample - Resazurin: a chemical that is purple when oxidized; colorless when reduced

Reductase Test - Resazurin will be added to milk; as milk is reduced by the bacteria, resazurin will turn from blue/purple to pink to colorless over time - I will add 1.0 mL resazurin to 10 mL high and how quality milk - Incubate in 37C water bath, check every 30 minutes to record time it takes for color to change and disappear

The Vaccine War Questionnaire 1. According to the documentary, approximately how many diseases are currently preventable by vaccination (as of 2010)? 16 2. Were any of the anti-vaxxers interviewed medical professionals? What kind of people were they (as in career/credentials)? No. The first anti-vaxxer was a parent. The second anti-vaxxer was celebrity woman Jenny McCarthy. The third anti-vaxxer was a man whose children was given 6 vaccines in 1 day and regressed. 3. One of the reasons the vaccine debate is still ongoing is due to anti-vaxxers “moving the goalpost” (changing their argument every time one is disproven). Name one of these “goalposts” that they use to blame vaccines. Goalpost 1: the MMR vaccine caused autism (12 studies show that is untrue) Goalpost 2: the idea thimerosal caused autism 4. Why is it so difficult to run scientific experiments to gather evidence on the efficacy of vaccines?

It is difficult due to ethics. There needs to be a control group (unvaccinated) and experimental group (vaccinated). 5. What is “herd immunity” and how does it relate to the importance of vaccination and the spread of disease? If most people in a population (herd) are immune to a particular disease, this form of immunity is referred to as herd immunity. The vaccinated people are protecting the vulnerable. This is important to vaccination because if not enough people are vaccinated then herd immunity decreases and the herd is at risk for spreading/contracting the disease. 6. What is one reason you could see symptoms of autism appear soon after vaccination, even if the vaccine doesn’t cause autism? a. Ingredients in the vaccines cause side effects that look like autism b. Autism symptoms usually first appear around the age when kids get vaccines c. There are no other reasons, when autism appears it is caused by the vaccines d. Children are emotionally affected from getting vaccinated and their behavior can be confused with autism 7. Some examples of diseases that have been basically eradicated in wealthy countries but could return if we don’t vaccinate properly are _______________ and _______________. a. Malaria b. Polio c. Tuberculosis d. Measles 8. What makes Denmark a great place to study if vaccines cause autism? Denmark is a great place to study vaccines because they collect demographic and health data routinely on all their citizens in a series of national registers (birth place, vaccines, when autism was diagnosed). 9. What were the findings from the Denmark studies in relation to the claims that the MMR vaccine causes autism and thimerosal causes autism? There was no difference meaning that children who did not get the vaccine

had the same risk of developing autism as those who did. Children given thimerosal before 1992 had identical autism rates as children given mercury free vaccines after that date. There was no association. 10. What are 2 reasons the general public distrust vaccines? - Misinformation - Mistrust of pharmaceuticals - Internet - Most public health officials...