lab practical #2
★Metabolic Testing of Bacteria (start at Introduction to Metabolic Testing)
1. Explain the purpose of studying metabolic tests and how a dichotomous key can help.
Metabolic tests set us up for unknown, the tests determine presence/absence of specific enzymes. The dichotomous key identifies the unknown.
2. Given experimental tubes or plates + any accessory materials, identify the specific test.
3. Review the Metabolic Charts and know: 1) the test name, 2) what you are specifically testing for, 3) the medium or substrate + any reagents used, 4) a positive vs. negative result and 5) the reason for a positive result.
4. Interpret the following abbreviations:
A acid, AG acid + gas, K alkaline, SIM Sulfide, Indole, Motility.
★Yeasts
1. Know that yeasts are eukaryotic fungi.
2. Identify budding yeast of 3. Identify yeast ascospores of
Saccharomyces cerevisiae under the microscope. Saccharomyces cerevisiae
Asexual Sexual
Shoe print
4. Know that certain yeasts can cause mycoses in immunocompromised people.
Fungal infections
5. Identify Candida albicans under the microscope.
Budding, asexual
Purple shoe prints
6. Review the Pathogens Chart for yeasts and know: 1) the official name (binomial nomenclature), 2) the name of the disease it causes, 3) the etiology, 4) the mode(s) of transmission and 5) prevention methods
★Molds
1. Know that molds are eukaryotic fungi.
2. Know the asexual life cycle of a mold, and identify the life stages under the microscope
spore germinate when conditions are beneficial
hypha, starts to grow, single-string
Mycelium, tangle of strings fuzzy mat
spore-producing body, makes spores, claw & puff ball
3. Know the name of the selective medium for molds and what is being selected.
Sabouraud agar, selective for fungi as high sugar, low pH = bacteria do not like
4. Identify under the microscope:
Penicillium and Rhizopus
Claw source of antibiotic puff ball (black) bread mold
5. Know that certain molds can cause mycoses in immunocompromised people.
Opportunistic fungal infection ex. Superficial: athlete's foot, ringworm…
Systemic↓
6. Identify under the microscope:
Aspergillus and Coccidioides
Brown spore-producing bodies (Valley fever)
7. Review the Pathogens Chart for molds and know: 1) the official name (binomial nomenclature), 2) the name of the disease it causes, 3) the etiology, 4) the mode(s) of transmission and 5) prevention methods.
★Plasmodial Slime Molds
1. Identify a slime mold.
Eukaryote, decomposers, free-living (not harmful)
Yellow, string-looking / blood-vessel like
2. Know the name of the slime mold that we used in lab.
Physarum polycephalum
3. Differentiate a plasmodium from a sclerotium.
Plasmodium: live, lives in moist places (forests), eats microbes & organic matter,
Sclerotium: dehydrated, not growing
4. Know the name of the medium for slime molds.
Tap water agar
5. Explain the purpose of cytoplasmic streaming.
Making / moving nutrients + oxygen from one part to another, all connected
★Unknowns
1. Know the steps you performed, in general, to identify your unknown.
Day1: gram stain, streak plate
Day2: described colony morphology, did dichotomous key & inoculated media
Day3: use key to identify unknown
2. Apply information learned to determine a species name given test results and a dichotomous key.
★Viruses
1. Know that viruses are acellular infectious agents.
2. Identify a Downey cell from infectious mononucleosis under the microscope→
3. Review the Pathogens Chart for viruses and know: 1) the official name (binomial nomenclature), 2) the name of the disease it causes, 3) the etiology, 4) the mode(s) of transmission and 5) prevention methods.
★Blood Smear
1. Identify the different leukocytes under the microscope.
2. Know the frequency of each leukocyte in blood.
Most to least, NLMEB
★Biotechnology
1. Explain the purpose of gel electrophoresis and how it works.
Separates DNA fragments by size; get dyed along the way, smallest run the fastest
2. Explain why a molecular weight ladder is used in gel electrophoresis.
Used as a size standard, identifies unknown
3. Explain the purpose of PCR and how it works.
Amplifies, copies a gene of interest over and over
★CRISPR-Cas Gene Editing
1. Explain the origin of the CRISPR-Cas system and how it can be used for gene editing.
Bacteria adaptive immune system to destroy bacteriophages, faster response 2nd time
Gene editing: disable bad gene or insert copy of a good gene
2. Explain the purpose of this experiment. To see if we can cut the lac Z gene
3. Identify the components of the two engineered plasmids.
pD: donor template DNA
pDG: “” and sgRNA (to cut DNA) guide
4. Explain how you introduced the plasmids into the host E. coli.
Artificial transformation, heat shock
5. Predict and observe gene expression on the four plates.
Not cut: blue colonies
Cut & repaired: white colonies
Cut & not repaired: no colonies
★Simulated Epidemic
1. Define:
Epidemiology, study of when / were diseases occur & how they are transmitted
Epidemic, spiked levels of disease in a population
Pandemic, global epidemic
2. Explain the purpose of this experiment. Simulate the spread of an epidemic within a population
3. Given a plate, identify the simulated epidemic experiment.
Orange colonies!!
4. Identify the bacteria used in this experiment and explain why they were chosen.
Sarcina aurantiaca = orange colonies, evidence of spread of disease.
White colonies could be anything / contamination
★Movie: Spillover-Zika, Ebola and Beyond
1. Describe the characteristics of Zika, Ebola and Nipah viruses, including: location(s), symptoms, transmission and prevention.
Zika: uganda, joint pain, fever, rash, microcephaly, mosquitos, bodily fluids, congenital, kill 🦟
Ebola: congo, headache, dehydration, internal bleeding, person-to-person, quarantine
Nipah: bangladesh, fever / loss of consciousness, neuro damage, saliva & bats (raw palm sap)
2. Besides Zika, name some other mosquito-borne diseases. Dengue, west nile virus…
3. Define “spillover,” and discuss how this is being promoted. Getting things from wild animals, promoted as we get more exposed w wild animals and their habitats
4. Define “zoonotic,” and name some examples. Disease that jumps species (animal to people)
Ex. bats (covid, ebola), birds (flu)
5. Define “contact tracer,” and describe how contact tracing can be helpful. Worker that tracks down people who came in contact w infected person eventually reaching patient zero
6. Discuss the goal of the PREDICT program. Identity next pathogen before se descontrole
7. Explain how we can effectively prevent global outbreaks of disease.
Early detection & ability to treat diseases
Proper care / resources for infected people and workers
★Throat Culture
1. Explain the purpose of this experiment and what you are testing for.
Looking for pattern hemolysis, testing whether bacteria is pathogenic or not
2. Know the name of the medium used. Sheep’s blood agar
3. Given plates, differentiate hemolysis types:
Alpha, partially cleared out, green, halo
Beta, complete clearing, Strep. or Staph (pathogenic)
Gamma, no clearing at all, normal microbiota
4. Know which type of hemolysis is considered pathogenic, and list examples of possible pathogens. Beta, streptococcus, staphylococcus
5. Understand the concept of a healthy carrier and how this impacts healthcare workers.
Shows no symptoms yet spreads infection around, concerning for immunocompromised people
★Urine Culture
1. Define:
Cystitis, bladder infection (lower urinary tract)
Pyelonephritis, kidney infection (upper urinary tract)
Dysuria, painful/difficult urination
Pyuria, WBC in urine pus
Hematuria, RBC in urine
2. Identify the most common source of urinary tract infections and the risk factors that promote development of disease.
Most common: Fecal microbiota (E. coli)
Risk factors: being female, catheters, holding urine…
3. Explain the purposes of this experiment.
Count how many bacteria / mL are in one sample (if # of bacteria is significant or not)
How many diff types of bacteria present
Whether or not (or both) are lactose fermenters
4. Know the name of the medium used for counting bacteria. Sheep’s blood agar
5. Know the name of the streak method and why it does not need to be precise.
Semiquantitative streak method, no need to be precise as # is either really low or high.
Low # = normal microbiota. (<10k) High # = infection (>100k bacteria/mL)
6. Know the name of the differential and selective medium, and specify what is being selected and differentiated.
McAnkey agar (pink). selected= gram (-). differentiated= lactose vs non-fermenters (white)
7. Given plates, differentiate lactose fermenters vs. non-lactose fermenters.
Pink colonies white colonies
★Oral Microbiota
1. Identify oral microbiota under the microscope (cheek cells and mixed bacteria).
Large cells, square-ish
2. Understand that bacteria are numerous and diverse in the mouth.
★Eukaryotic Parasites
1. List the three categories of eukaryotic parasites.
1. Protozoa 2. Helminths 3. Artropodes
Amoeba, flagellates, WORMS, nematode, INSECTS, ARACHNIDS
ciliates, sporozoans cestode, trematode ^6 legs ^8 legs
2. Differentiate between a mechanical and biological vector, and provide examples of diseases vectored by mosquitoes and ticks.
Mechanical, physically carries pathogen externally, ex. Fly carrying e. coli from feces
Biological, carries pathogen internally, vital part of its life cycle
Mosquito: Malaria, Zika, Dengue…
Tick: Lyme disease…
3. Identify all of the eukaryotic parasites under the microscope.
4. Review the Pathogens Chart for eukaryotic parasites and know: 1) the official name (binomial nomenclature), 2) the name of the disease it causes, 3) the etiology, 4) the mode(s) of transmission and 5) prevention methods.
★Staphylococcus aureus Rapid Test
1. Explain the purpose of running this rapid test. Diagnose quickly!
Common: skin & wound infections
Other infections: sepsis, pneumonia, endocarditis…
Can cause toxic shock syndrome & necrotizing fasciitis
2. Given test materials, identify the Staphylococcus aureus passive agglutination test, and determine if test results are positive or negative.
Looking for Ag, latex beads are coated with Ab
(+)=clumping
3. Explain how this test works in terms of antigen and antibody interactions.
Antibodies react w antigen if present
★Streptococcus pyogenes Rapid Test
1. Explain the purpose of running this rapid test. Diagnose quickly!
Common: strep throat
Others: sinusitis, pneumonia, scarlet fever…
Immunologic disorders: rheumatic fever & glomerulonephritis
Can cause toxic shock syndrome & necrotizing fasciitis
2. Given test materials, identify the Streptococcus pyogenes modified ELISA test, and determine if test results are positive or negative. Color change!
Looking for Ag
Stick coated w Ab
(+)= 2 red lines, (-)= 1 red line, (no lines)= not working
3. same
★The Five-Second Rule
1. Explain what the five-second rule states. Safe to eat food that's fallen if its only 5 s
2. Discuss the risks of eating food dropped on the floor. Pathogens may attach, food-born illness
3. Discuss reasons why people eat food that has been dropped on the floor.
Ignore risk, highly desired food, no previous issues
4. Explain the purpose of this experiment. See if this rule is valid
5. Given experimental plates, identify the five-second rule experiment.
Nutrient agar plates, more bacteria in After plate. Wet food attracts more bacteria than dry
6. Discuss your results rule is not true
★Antibiotic Sensitivity
1. Explain the purpose of this experiment.
See which bacteria is sensitive or not to a given antibiotic
2. Understand the basic setup of the Kirby-Bauer disk diffusion test and how it works.
Spread bacteria out on 2 plates → antibiotic disc → diffuses → kills/not bacteria
Clear area = zone of inhibition
Also, Pseudomonas aeruginosa turns green due to green pigment made by the bacteria
3. Interpret zones of inhibition as being sensitive, intermediate or resistant. SIR
4. Explain why a bacterial colony might grow in a zone of inhibition.
Resistant mutants
5. Explain what an E test adds.
MIC; minimum inhibitory concentration
Lowest dose of antibiotic that still works