Practical Lab Final Flashcards
Define ocular lens (what is it called, what does it do, and what is its magnification?)
Binocular lens. Allows both eyes to focus on the specimen. Has a magnification of 10x.
Define ocular adjustment
Allows you to adjust each separate ocular eyepiece for people who have differences between their eyes
Define revolving nosepiece/ turret
Holds objective lenses. Rotation of this allows the objective lenses to ‘click’ into place for viewing
1) Define objective lenses.
2) What magnifications are they?
3) What total magnifications do they provide?
1) The lenses on the revolving turret. Provide the major magnification of the microscope.
2) 4x, 10x, 40x, 100x.
3) The ocular lens (10x) times the magnification (ex: 40x * 10x = 400x total)
1) Where is a slide placed on a microscope?
2) What holds the slides in place for observation?
3) What knob moves the stage to view different parts of the slide?
1) The stage
2) The stage clips/ slide clamp
3) The stage adjustment
1) What is the series of lenses that focuses the light traveling from the light source onto the specimen and allow for bright field microscopy?
2) What expands or reduces the amount of light hitting the specimen?
3) What is a power control that adjusts the power from the light source? What does higher power equal?
1) Condenser
2) Iris diaphragm
3) Rheostat; higher power = brighter light. Ranges 0-10.
1) What is the light bulb on a microscope called?
2) What type of focus should only be used on lower power objectives (because it moves the stage in large increments)?
3) What type of focus can be used on higher power objectives?
1) Light source
2) Course focus
3) Fine focus
What turns the microscope on and off?
Power switch
Gram stain:
1) What color are gram + species? Give examples.
2) What color are gram - species? Give examples.
1) Purple. Ex: Staphylococcus aureus & S. epidermidis, B. subtilis and B. cereus.
2) Red. Ex: E. coli, Salmonella enteritidis, S. sonnei, K. pneumoniae, P. vulgaris.
1) What color are bacteria on an endospore stain? What color are endospores?
2) Acid fast stain: What color are acid-fast bacteria? Why? What color are non-acid-fast bacteria? Why?
1) Red; endospores are green
2) Acid-fast: red (carbol fuchsin); non-acid-fast: blue (methylene blue)
1) What is a statistically valid plate count?
2) How do you count the number of cells on a plate?
3) What is the equation for colony count?
1) Between 30 and 300 colonies
2) #colonies / TDF = # of cells
3) #cells per mL X TDF = colony count
1&2) What are the two methods used to generate a bacterial growth curve in lab?
3) What are the 4 phases of a bacterial growth curve?
1) A viable plate count and 2) turbidity measurement using a Nephlo flask and a spectrophotometer
3) Lag, log, stationary, and death
Which two enzyme tests required a stab inoculation?
Gelatinase and SIM
Which 3 enzyme tests were streaked for isolated colonies?
1) SM110
2) MS
3) TSA
Why does the lysine test require mineral oil?
To make the broth an anaerobic environment to force the culture to ferment glucose
Which 3 enzyme tests required a loopful into broth?
1) Nitrate
2) Coagulase (TSB)
3) Urea
Which 2 enzyme tests required a line inoculation?
1) DNase
2) Starch (amylase)
Name 5 tests that differentiate between S. aureus and S. epidermidis
1) MSA
2) BA
3) SM-110
4) Coagulase
5) DNase
What 2 tests differentiate between BS and BC?
1) MSA
2) LM
Name 2 tests useful in the identification of the genus Proteus
1) PHE
2) Urea
Which tests should be used for G+ rods? (1)
LM (litmus milk)
Which tests should be used for G+ cocci? (5)
1) BA
2) MSA
3) Coagulase (SA+; SE-)
4) SM-110
5) MS
Which tests should be used for G- rods (6)?
1) SS
2) MAC
3) TSI
4) Citrate (EC-; EA+)
5) VP (EC-; EA+)
6) Methyl Red (EC+; EA+)
Which tests should be used for G- cocci? (1)
1) SIM
Which tests should be used for G- bacteria in general? (1)
EMB
Which tests are differential only? (6)
1) LM
2) TSI
3) Citrate
4) VP
5) MR
6) SIM
Which tests are selective only? (2)
Coagulase and BA
Which tests are both selective and differential? (6)
1) SS
2) SM-110
3) MAC
4) MSA
5) MS
6) EMB
List the enzymes the following tests look for the presence of, and state what each enzyme degrades:
1) DNase
2) Urea
3) Nutrient starch
4) LYS
5) Nutrient gelatin
6) Catalase
7) PHE
8) ONPG
9) Coagulase
10) MS
1) DNase: DNase, DNA
2) Urea: Urease, urea
3) Nutrient starch: Amylase, starch
4) LYS: Lysine decarboxylase, amino acid
5) Nutrient gelatin: Gelatinase, gelatin
6) Catalase: Catalase, H2O2
7) PHE: Phenylalanine deaminase, phenylalanine
8) ONPG: B-galactosidase; lactose fermentation
9) Coagulase: Coagulase, protein clotting
10) MS: Saccharase; sucrose
What question does a NO3 (nitrate) test answer?
Does bacteria use NO3- as a terminal electron acceptor in anaerobic respiration (and denitrify it to N2)?
1) What does a SIM test test for? (3 things)
2) What does an EMB test test for? (2 things)
1) SIM: Motility and H2S to FeS production (some G- cocci); indole production (from tryptophan breakdown) (EC+; EA-)
2) EMB: Ability to grow on EMB’s eosin Y & methylene blue (G-) and coliform differentiation (by color and lactose fermentation)
1) What does an SM-110 test test for? (3 things) What bacteria are positive?
2) What does an MS test test for? What bacteria are positive?
1) Ability to grow on NaCl (G+); gelatinase presence; glycolysis of fructose-phosphate from mannitol (G+ cocci: SA+; SE-)
2) Ability to grow on MS’s sucrose due to saccharase (G+ cocci: SM and EF+)
What do the following tests look for?
1) Methyl red
2) VP
3) Citrate
1) Ability to ferment (make) mixed acids, decreasing pH (EC+, EA-)
2) Ability to ferment alcohol, creating intermediate acetylmethylcarbinol (EC-; EA+)
3) Ability to grow on citrate which releases ammonia, increasing pH (EC-; EA+)
What does a TSI test look for? (3 things)
Include the color of each thing
1) Ability to ferment glucose (red over yellow, K/A)
2) Ability to ferment lactose and/or sucrose (all yellow, A/A)
3) H2S production (black)
What does an MAC test look for? (2 things)
Include what species are positive for each thing.
1) Ability to grow on MAC’s CV and bile salts (G-)
2) Ability to ferment lactose (EC+, red)
What does an SS test look for? (3 things).
Include what species are positive for each thing.
1) Ability to grow on SS’s bile salts (G- non-coliform rods)
2) Ability to ferment lactose (EC+, red). Neutral red indicator
3) H2S production (Salmonella+)
What does an MSA test look for? (2 things)
Include what species are positive for each thing.
1) Ability to grow on MSA’s high NaCl (G+ cocci: Bacillus and staphylococcus)
2) Ability to ferment mannitol (SA+)
1) What genuses are a BA test differential for?
2) What question does a BA test answer?
Include which species are positive.
1) Staph and strep
2) Does bacteria do gamma, alpha, or beta hemolysis? (G+ cocci: SA does beta; SE does gamma)
What 2 things does a LM test look for? Include what species is positive.
1) Ability to ferment lactose and produce acid, decreasing pH (Bacillus)
2) Ability to break down casein protein in milk
1) What 2 tests use neutral red indicator?
2) What color is neutral red indicator when introduced to an acid?
1) SS and MAC
2) Red: acidic
1) What 3 tests use phenol red indicator?
2) What color is phenol red when introduced to an acid?
1) MSA, TSI, Urea
2) Yellow: acidic
1) What test uses litmus milk as an indicator? What color is it when positive?
2) What test uses methyl green as an indicator? What color is it when positive?
3) What test uses bromcresol purple as an indicator?
4) What test uses bromothymol blue as an indicator?
1) LM: litmus milk (white: acidic = +)
2) DNase: methyl green (clearing = +)
3) LYS: bromocresol purple (purple/black: basic = +)
4) Citrate: bromothymol blue (vivid blue: basic = +)
Which 4 tests can help differentiate between Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE)?
Report when each is positive and negative
1) Coagulase (TSB): SA+; SE-
2) BA: SA beta; SE gamma
3) MSA: SA+; SE-
4) SM-110: SA+; SE-
Salmonella and shigella grow well on which two media?
MAC and SS
1) What two bacterial species ferment mannitol (MSA test)? What color does this result in?
2) What two bacterial species do not ferment on MSA? What color does this result in?
1) S. aureus (SA) and B. subtilis (BS): turn yellow (acidic)
2) S. epidermidis (SE) and B. cereus (BS): stay red (basic)
What 7 media can help differentiate between E. coli (EC) and E. aerogenes?
1) VP: EC-; EA+
2) Citrate: EC-; EA+
3) Indole: EC+; EA-
4) Methyl red: EC+; EA-
5) MAC: EC+; EA-
6) SS: EC+; EA-
7) SIM: EC+; EA-
List the 3 media that use phenol red and what color they are when positive
1) MSA: Turns yellow when positive
2) TSI: Red is ‘k’, yellow is ‘a’
3) Urea: Turns red when positive
Name the 4 antiseptics/ disinfectants used in the bacterial death exercise
1) Clorox (QAC)
2) Pinesol (DET)
3) Greenworks (CIT)
4) Iodine (IO)
What type of disinfectant/ antiseptic is clorox?
A quaternary ammonium compound
What type of disinfectant/ antiseptic is pinesol?
A detergent
What is greenworks?
Citric acid
What two characteristics would you look for in a test regarding the efficacy of chemical agents on bacteria?
1) Quantitative
2) Reproducible
What 3 categories of techniques were used to kill bacteria in the bacterial death exercise?
1) Disinfectants/ antiseptics
2) Antibiotics
3) UV radiation
What 4 categories antibiotics were used to inhibit bacterial growth in the bacterial death lab? What antibiotic was used to represent each category?
1) Nucleic acid inhibitor: RIF
2) Cell wall inhibitor: AMP
3) Protein synthesis inhibitor: TET
4) Structural analog: SXT
1) What type of test was used to measure the efficacy of disinfectants/ antiseptics and antibiotics on bacteria?
2) How is this test interpreted?
1) Disk diffusion test
2) If there is an area of no growth around the disc, the agent was effective
How many seconds of UV radiation did each condition get in the bacterial death lab?
0, 10s, 30s, 1m, 2m, 5m, 10m
1) What was the first test used to measure the efficacy of chemical agents on bacteria? Why was it not preferable?
2) What test replaced that test?
3) What test replaced the second test?
1) Broth dilution testing; time consuming and resource intensive
2) Disk diffusion testing
3) E-test strips
What are the steps of a disk diffusion test?
1) Streak a plate to create a lawn of bacteria
2) Apply antibiotic or disinfectant disk
3) Incubate
4) Zone of inhibition is created around the disk if agent is effective (i.e. no bacteria around disk)
Why is there a gradient observed around the disk in the disk diffusion test?
Because the agent diffuses from the disk into the agar medium
Why did the BS 5-day old culture grow better than the newer culture under UV conditions?
Because the bacteria had sporulated
Is UV radiation more effective against G+ or G- bacteria? Why?
More effective against G- bacteria; G+ bacteria that can form spores are more protected
Name 5 environmental conditions that can influence bacterial growth
1) Oxygen
2) Osmotic pressure
3) pH
4) Temperature ranges
5) Liquid water presence
1) What one species (that we work with) clarifies litmus milk?
2) What species is a denitrifier?
3) What 2 things makes P. vulgaris unique?
1) B. subtilis
2) Ps. aeruginosa
3) It makes an alkaline broth in urea and breaks down phenylalanine to make PPA
1) How does a Brewer jar work?
2) How does a candle jar work?
1) They become anaerobic by using a GasPak; the Pak generates H2 and CO2; the H2 reacts with the oxygen in the jar to form water vapor by the use of a catalyst.
2) A lit candle is added to a jar and tightly sealed; the flame makes CO2 and extinguishes itself when the O2 concentration is too low (6-8%).
1) Name 3 ways to enumerate (count) bacteria
2) What is one way to estimate bacteria count?
1) Viable plate count, MPN, and turbidity measurements using a flow cytometer
2) Spectrophotometer to measure turbidity.
1) How can you test to see if someone has S. aureus on their skin or throat?
2) How can you test to see if someone has yeast on their skin?
1) MSA plate (turns yellow)
2) SDA (sabouraud dextrose agar)
What two places of the body were microbes isolated from?
1) The skin (behind the ear)
2) The oral cavity (back of the throat)
What plates were inoculated with the oral cavity swab?
1) TSA
2) BA
3) MSA
What plates were inoculated for the skin (behind ear) swab?
1) TSA
2) MSA
3) SDA
Why was the SDA plate used for the skin sample?
To isolate any yeast growing on skin
Name 2 types of bacteria that typically colonize the skin (exterior)
1) Micrococcus
2) Staphylococcus (aureus and epidermidis)
Name the 3 typical mucosal (mouth) colonizers
1) Staph aureus
2) Streptococci
3) Enterococcus
4) Corynebacterium
Where are bacteria typically abundant in the oral cavity?
Underneath the gums
1) What are the yellow bacteria typically isolated from the skin?
2) What are the white bacteria isolated from the skin?
1) Micrococcus: Yellow
2) S. epidermidis and S. aureus: White
Name the 3 bacteria typically seen in the mouth
1) Streptococci (tiny colonies)
2) Haemophilus
3) Cyanobacterium
What does clearing on a plate of isolated mouth bacteria indicate?
Beta hemolysis, which means it’s more likely a pathogen
1) What commensal microbes live on the skin?
2) What commensal microbes live in the mouth?
1) Staphylococcus, Micrococcus, Corynebacterium, Pityrosporum (yeast)
2) Staphylococcus, Micrococcus, Streptococcus
Name 5 potential pathogens
1) S. aureus
2) St. pyogenes
3) Neisseria
4) Haemophilus
5) S. epidermidis
1) What is agar?
2) What should bacterial media contain?
1) Carrageenan (seaweed)
2) Major elements (CHONPS), minor elements (Fe,Ca,K, Mg), trace needs (vitamins), an energy source, buffering capacity
Differentiate between complex and defined media
1) Complex: major constituents are organic plant or animal components (ex: TSB); exact nutritional amounts are unknown. Grow many types of bacteria.
2) Defined: synthetic media that contain precisely measured components. Generally grow a narrow range of bacteria.
How does autoclaving work?
High pressure saturated steam at 121c works because moist heat kills:
1) Increases motion of particles
2) Cleavage of H bonds between proteins
3) H2O molecules in steam are highly energized.
4) More penetration into liquids/ surfaces
Name 3 reasons why aseptic technique is important
1) Safety (of you)
2) Sterility (of substances)
3) Purity (of cultures)
Name and describe 3 ways in which we isolated cultures
1) Streak plate isolation: streak for isolated colonies
2) Pour plate isolation: dilute bacterial samples multiple times prior to pouring into molten agar.
3) Spread plate isolation: dilute bacterial samples multiple times prior to streaking a plate.
1) What test was created due to a problem with water contamination? When is it positive?
2) How are the results calculated?
1) Phenol red lactose fermentation. Yellow tube = positive.
2) MPN (most probable number) (number of positive tubes + chart).
How do you calculate the resulting dilution found on a plate?
TDF of last tube x amount transferred to plate
How do you calculate the number of bacteria per ml in each tube of a dilution? (if you’re given the original culture’s cell count)
Last tube’s number of cells/ml x SDF
How do you find the dilution used at each step of a dilution scheme? (the ratio)
Amount transferred: Amount already in tube + amount transferred
1) How else can 1:100 be written?
2) How else can 1:10 be written?
3) How else can 1:2 be written?
1) 10^-2
2) 10^-1
3) 5 x 10^-1
1) How do you calculate number of cells?
2) How do you calculate number of colonies?
1) #colonies / TDF
2) #cells per mL x TDF
1) What 2 types of bacteria are acid-fast stains best for?
2) Why are acid-fast stains used on these bacteria?
1) Genus Mycobacterium or Norcardia
2) These cells have a waxy component in their cell walls composed of mycolic acid, which makes them resistant to penetration by aqueous- based stains
1) What are the 2 reasons for heat-fixing and air-drying bacteria?
2) What two things does a gram stain tell you?
1) Kills the bacteria and makes sure they adhere to the slide
2) Shape and Gram + or -
Name and describe the 4 stages of differential stains
1) Primary stain: initial staining of all cells
2) Mordant: chemical reaction that anchors primary stain to one cell type
3) Decolorizer: chemical reaction that selectively removes primary stain
4) Counter stain: stains the cells that lost the primary stain
