microbio practical 2 exam

list the PPE you need to put on when working in the microbiology lab

lab coat, lab goggles, gloves,

describe the proper handling of your cell phone, food, drinks and cosmetics in the lab

no cell phones, food, drinks, or cosmetics in the lab

describe the proper clothing to wear in the lab as well as the safe ways to wear your hair and jewelry 

wear pants, close-toed shoes, hair tied up, avoid dangling jewelry

describe when you need to disinfect the benches

before and after the lab

focus a slide with bacteria under 100X objective lens using the compound microscope AND immersion oil within 10 minutes. 

• Start with the 4× then 10× then 40× objectives to locate the specimen

• Rotate to the 100× oil-immersion objective

• Place a drop of immersion oil directly on the slide

• Focus the 100× objective using only the fine-focus

• Only use immersion oil with the 100× objective, clean the lens with lens paper after use

identify accurately Gram-positive and Gram-negative bacteria on a microscope slide

• Gram-positive bacteria: PURPLE/VIOLET (they have a thick peptidoglycan cell wall that retains the crystal violet-iodine complex even after decolorization)

• Gram-negative bacteria: PINK/RED (their thin peptidoglycan layer and outer membrane lose the crystal violet during decolorization; they pick up the safranin counterstain)

identify accurately the shape and cell arrangement of cells on a microscope slide

• Shapes: Cocci (spherical), Bacillus (rod)

• Arrangements: Chains, clusters

catalase test substrate

hydrogen peroxide (H₂O₂)

catalase test enzyme

catalase (produced by bacteria)

catalase test products/results

• Products: Water (H₂O) and Oxygen gas (O₂)

• Positive result: Bubbling when H₂O₂ is added to the culture

• Negative result: No bubbles

identify an antibiotic inhibition zone obtained by the Kirby-Bauer method

The zone of inhibition is the clear area around an antibiotic disk where bacterial growth is absent on a Mueller-Hinton agar plate. The disk releases the antibiotic outward in a gradient. Bacteria unable to grow near the disk are susceptible; those growing close to or up to the disk are resistant.

accurately measure an inhibition zone of an antibiotic disk AND determine the bacteria's reaction to it - susceptible, resistant, or intermediate using an antibiotic chart

• Measure the diameter of the clear zone in millimeters using a ruler

• Then compare to the standardized chart for the specific antibiotic:

  • Susceptible (S): zone diameter ≥ susceptible breakpoint

  • Intermediate (I): zone diameter falls between S and R breakpoints

  • Resistant (R): zone diameter ≤ resistant breakpoint

describe the difference between alpha, beta, and gamma hemolytic bacteria AND be able to determine the hemolytic activity of a pathogen on a plate or a picture

• Alpha (α) hemolysis: PARTIAL hemolysis of red blood cells. Appears as a GREENISH or brownish discoloration around colonies on blood agar. The RBCs are not fully lysed but the hemoglobin is oxidized.

• Beta (β) hemolysis: COMPLETE hemolysis. Appears as a CLEAR, transparent zone around colonies — all RBCs are fully lysed.

• Gamma (γ) hemolysis: NO hemolysis. No change in the blood agar around colonies; agar appears unchanged (red/pink).

identify the basic characteristics of Enterobacteriaceae sp.

• Gram stain reaction

  • Gram-NEGATIVE

• Cell morphology

  • Bacilli (rods)

• Oxidase reaction

  • NEGATIVE 

• Glucose fermentation

  • POSITIVE (facultative anaerobes — ferment glucose)

list the selective and differential agents in MacConkey media

• Selective agents: Crystal violet and bile salts → INHIBIT Gram-positive bacteria; only Gram-negatives grow

• Differential agent: Lactose + neutral red indicator → differentiates lactose fermenters from non-fermenters

be able interpret the results about growth and lactose fermentation accurately (MacConkey agar)

• Results:

  • Lactose fermenter (e.g., E. coli): PINK/RED colonies (acid production lowers pH → neutral red turns pink)

  • Non-lactose fermenter (e.g., Salmonella): YELLOW colonies

  • No growth: Gram-positive organism

know how to distinguish positive from negative reaction for the oxidase test

• positive: dark blue/purple at end of q-tip 

• negative: no color change

carbohydrate fermentation substrate(s), type of reaction, product(s)

• substrates: glucose or lactose

• type of reaction: fermentation of sugar

• products: pyruvic acid, H₂ and CO₂ (gas)

urease substrate(s), enzyme/type of reaction, product(s)

• substrate: urea

• enzyme: urease

• products: CO₂ + ammonia

citrate substrate(s), enzyme/type of reaction, product(s)

• substrate: citrate

• type of reaction: citrate utilization as sole carbon source

• product: alkaline products

PADase substrate(s), enzyme/type of reaction, product(s)

• substrate: phenylalanine

• enzyme: phenylalanine deaminase

• product: phenylpyruvic acid

desulfurase substrate(s), enzyme/type of reaction, product(s)

• substrate: sulfur containing compounds

• enzyme: desulfurase

• product: hydrogen sulfide (H₂S) gas

tryptophanase substrate(s), enzyme/type of reaction, product(s)

• substrate: tryptophane

• enzyme: tryptophanase

• product: indole

motility substrate(s), enzyme/type of reaction, product(s)

• no specific substrate/product

• type of reaction: whether bacterium is motile (has flagella)

name the three bacterial properties that are tested by the SIM tube

1. Sulfide (H₂S) production — desulfurase activity

2. Indole production — tryptophanase activity

3. Motility — flagella presence

list the three sugars which fermentation is tested in TSI (triple sugar iron agar)

1. glucose

2. lactose

3. sucrose

describe how carbohydrate fermentation result is interpreted

• positive (acid produced): yellow color

• positive (gas produced): gas bubble trapped in Durham tube

• negative: red = no fermentation, pink/magenta = alkaline shift

describe how urease result is interpreted

• positive: hot pink color → ammonia turned medium pH to alkaline

• negative: yellow/no color change

describe how citrate result is interpreted

• positive: blue color + visible growth → pH rises, bromothymol indicator changes color

• negative: green/no color change + no growth 

describe how PADase result is interpreted

• positive: dark green color → ferric chloride reacts with phenylpyruvic acid

• negative: yellow/no color change

describe how desulfurase result is interpreted

• positive: black precipitate along stab line → H₂S reacts with iron in the media to form iron sulfide

• negative: no black color

describe how tryptophanase result is interpreted

• positive: red ring at top → tryptophane reduced to indole 

• negative: yellow ring/no color change (no indole produced)

describe how motility result is interpreted

• positive: cloudy growth spreading away from stab line (motile)

• negative: growth only along stab line (non motile)

identify the basic characteristics of Bacillus sp.

• Gram stain reaction (type of cell envelope)

  • gram-positive

• Cell morphology and cell arrangement

  • rods, chains

• Catalase status

  • POSITIVE

• Oxygen requirements for growth

  • AEROBIC

• Endospore formation

  • positive

• Natural common habitats

  • soil, water, oxygen-rich environments

identify the basic characteristics of Clostridium sp.

• Gram stain reaction (type of cell envelope):

  • gram-positive

• Cell morphology and cell arrangement

  • rods, chains

• Catalase status

  • NEGATIVE

• Oxygen requirements for growth

  • ANAEROBIC

• Endospore formation

  • positive

• Natural common habitats

  • animal GI tracts, oxygen-free environments

describe diseases caused by B. anthracis – signs and symptoms, mode of transmission, treatment, prevention, and vaccine (if available)

• Pulmonary anthrax

  • Signs & symptoms: cough, shortness of breath, chest pain, fever, fatigue 

  • Mode of transmission: inhalation 

  • Treatment: ciprofloxacin or doxycycline

  • Prevention: avoid infected animal product exposure 

  • Vaccine: AVA (anthrax vaccine adsorbed)

• Cutaneous anthrax

  • Signs & symptoms: non-painful black lesion 

  • Mode of transmission: cut in the skin

• Intestinal anthrax

  • Signs & symptoms: nausea, vomiting, diarrhea

  • Mode of transmission: ingesting contaminated food

describe diseases caused by B. cereus – signs and symptoms, mode of transmission, treatment, prevention, and vaccine (if available)

• Food poisoning

  • Signs & symptoms: nausea and vomiting, diarrhea  

  • Mode of transmission: ingesting contaminated food 

  • Treatment: supportive care

  • Prevention: proper refrigeration of food

describe diseases caused by C. perfringens – signs and symptoms, mode of transmission, treatment, prevention, and vaccines (if available)

• Gas gangrene 

  • Signs & symptoms: rapidly spreading tissue necrosis, gas bubbles in tissue, severe pain, systemic shock

  • Mode of transmission: spores enter through deep wounds

  • Treatment: surgical debridement, penicillin

describe diseases caused by C. tetani – signs and symptoms, mode of transmission, treatment, prevention, and vaccines (if available)

• Tetanus (lockjaw)

  • Signs & symptoms: severe muscle spasms in the jaw, respiratory paralysis 

  • Mode of transmission: spores enter through deep wounds

  • Treatment: tetanus immune globulin (TIG), penicillin

  • Vaccine: DTaP/Tdap

describe diseases caused by C. botulinum – signs and symptoms, mode of transmission, treatment, prevention, and vaccines (if available)

• Botulism 

  • Signs & symptoms: muscle paralysis, weakness, lethargy, difficulty eating, possible respiratory failure 

  • Mode of transmission: ingesting contaminated food

  • Treatment: heptavalent antitoxin (HBAT), supportive care

  • Prevention: proper canning techniques, never give honey to infants 

describe how anaerobic chambers work and their purpose

• Used to grow Clostridium sp.

• Gas-Pac releases CO₂ and H₂ → H₂ binds to O₂ creating water and removing it from the chamber → creates anaerobic environment for the bacteria

describe how the sodium thioglycolate test works

• Used to determine an organism’s oxygen requirements

• Inoculation of a species in a medium with sodium thioglycolate and L-cysteine → two compounds reduce O₂ and create low oxygen environment at bottom of the tube → thioglycolate tube is inoculated with a bacterial loop → after inoculation Clostridium sp. will grow closer to the bottom in the reduced oxygen concentration

describe how the starch plate test is used to test for amylase and the role of iodine

• Used to identify Bacillus sp.

• Starch plate is incubated with bacteria and allowed to grow → plate is flooded with iodine → iodine binds to starch when present, turning the agar blue → if bacteria produces amylase, starch around the growth will get digested

interpret results for amylase test correctly

• Amylase-positive: clear (colorless) halo surrounding a colony; larger halo = more enzyme activity

• Amylase-negative: No halo (blue-grey right up to colony edge)

interpret results for sodium thioglycolate test correctly

• Aerobe: Growth ONLY at the top (requires O₂)

• Anaerobe: Growth ONLY at the bottom (killed by O₂)

identify the basic characteristics of Chlamydia trachomatis

• Gram stain reaction (type of cell envelope)

  • Gram negative spheres/ovals

identify the basic characteristics of Neisseria gonorrheae

• Gram stain reaction (type of cell envelope)

  • Gram negative cocci

describe diseases caused by Chlamydia trachomatis

• Intracellular

• Signs & symptoms: often asymptomatic, urogenital pain during urination/sexual intercourse, discharge, abdominal pain, vaginal bleeding, fever, trachoma 

• Mode of transmission: Direct contact with infected mucosal surfaces (sexual contact)

• Treatment: antibiotics (azithromycin, doxycycline, erythromycin), recommended for partners too 

• Prevention: barrier methods of protection (condoms)  

• Complications if untreated: pelvic inflammatory disease (PID) → infertility or ectopic pregnancy, inflammation of testicles → sterility, passed from mother to newborn

describe diseases caused by Neisseria gonorrheae

• Extracellular

• Signs & symptoms: often asymptomatic, similar to chlamydia; pain during urination/intercourse, increased discharge, vaginal bleeding between periods, infection in the eye, mouth (sore throat), rectum 

• Mode of transmission: sexual contact 

• Treatment: single intramuscular injection of antibiotic ceftriaxone, recommended for partners too

• Prevention: barrier methods of protection (condoms), antibiotic eye drops for newborns 

• Complications if untreated: passed from mother to newborn, infection can progress to the joints and heart 

• Concerns about antibiotic resistance: antibiotic resistance in Neisseria gonorrheae has been steadily increasing 

describe how NAATs are used to identify pathogens

• Gold-standard diagnostic method for both C. trachomatis and N. gonorrhoeae

• Able to detect a single gene from a pathogen and amplify it to about a billion copies → copies can be detected → infection will be diagnosed even in asymptomatic patients 

describe how PCR works and describe the role of each component – primers, template DNA, buffer, enzyme, dNTPs

• PCR (Polymerase Chain Reaction) amplifies a specific DNA sequence exponentially. Each cycle roughly doubles the target sequence. After ~30-40 cycles, you go from a few copies to billions.

  • Primers: small pieces of DNA which match part of the sequence of a specific gene and are needed to start the replication of said gene 

  • Template DNA: sample from which your fragment will be amplified

  • Buffer: provides optimal conditions for the enzyme 

  • Enzyme: DNA polymerase linking the nucleotides and making the new DNA 

  • dNTPs: new DNA – mixture of nucleotides with the four types of bases A, T, G, C

describe the outcome of a PCR reaction if a component is omitted from the reaction

NO product/poor product produced

describe the function of the three main steps in a PCR cycle

• Denaturation

  • Temperature is brought up high to 95º C for 2 min

  • Temp is high enough to break down weak hydrogen bonds between complementary strands and separate them 

  • Not high enough to break the covalent bonds holding together the nucleotides in each strand 

• Annealing

  • Temperature gets lowered to ~50º C (exact temp varies for each PCR reaction)

  • Since DNA strands were separated earlier, the primers will be able to anneal to the template DNA as the temperature is lowered

• Elongation 

  • Temperature is raised to 72º C which allows the DNA polymerase to bind to the primers and build the new DNA strand 

  • DNA molecule is duplicated → PCR cycle is repeated for 30-40 cycles and creates about a billion copies of the gene

understand how to estimate the size of an unknown DNA piece using the DNA ladder on a gel or a picture of a gel

1. Identify and label the ladder bands with their known sizes.

2. Locate your unknown band in the gel lane.

3. Find which two ladder bands it falls BETWEEN.

4. Estimate size by interpolation: if the unknown band is between the 500 bp and 1000 bp bands and appears roughly halfway, estimate ~750 bp.

be able to determine if a patient is negative or positive for a pathogen based on the PCR results from a NAAT test

• PCR amplifies a specific DNA fragment if the pathogen is PRESENT

• Positive result: A BAND appears in the patient's lane at the expected size (matching the positive control band size, confirmed with the ladder)

• Negative result: NO band appears in the patient's lane (no target DNA was present to amplify)