Microbiology Lab Practical 1 Flashcards
What are the general nutritional requirements for growth?
- water
- chemical element
The 4 physical factors that affect growth of an organism?
- salt and sugar concentration
- temperature
- oxygen gas
- pH
What ingredient is used as a solidifying agent in media?
agar
What type of organism makes up agar media?
algae
what percentage of organisms can be cultured in an artificial (laboratory) setting?
1 percent
By what process do most bacteria divide?
- binary fision
The temperature (in celsius) for the the freezer is?
-20 degrees
The temperature (in celsius) for refrigerator is?
4-10 degrees
The temperature (in celsius) for room temperature is?
20-25 degrees
The temperature (in celsius) for body temperature is?
37 degrees
What percentage of organisms require oxygen for growth?
50%
Define negative control?
- no growth
Define positive control?
- expect growth
Why might an organism fail to grow?
- none made it alive, dies before
- culture is bad
- physical conditions aren’t correct
Why might two different organisms show exactly the same growth features even if they aren’t supposed to?
- cross contaimination
what is a meniscus?
- the concave appearance artifact
What growth patterns are similar or different among the organisms tested?
- bacillus only had a pellicle
- staph/e.coli were both turbid
Is it possible for an organism to produce more than one growth pattern in the same tube?
yes
A film growth on the surface is a what?
pellicle
Even cloudiness is called what?
uniform fine turbidity
Growth adhering to the lass at the liquid/air interface is called what?
a ring
Suspended flakes or particles are called?
flocculent
A layer of growth at the bottom of the broth is called?
sediment
How do you calculate total magnification?
objective x ocular = total magnification
If a student wants to learn morphology, arrangement and motility of an organism what type of preparations would they use?
- wet mount preparation or the hanging drop procedure
Dyes that are positively charged are attracted to which components of cells?
- negative components of living cells
What charged dye will cause the cell to pick up the color?
- positively charged dye
Dyes that are negatively charged are attracted to what charge components?
- positive components
What is a simple stain?
- a procedure that uses one positively charged dye to give cells color
What are some examples of simple stains?
- methylene blue, crystal violet, safranin, and manevals
What a negative stain?
- a procedure that uses a negatively charged dye to stain the background while the cells remain virtually colorless
What are some examples of negatively charged acidic dyes?
- eosin, india ink, migrosin and congo red
What is morphology?
- shape
What are some advantages of making a heat fixed smear?
- kills the organisms
- organisms adhere to the slide
- organisms take up the stain more readily
What are some disadvantages of making a heat fixed smear?
- distorts the true size of the cell
- may destroy heat- sensitive cell structures
- may create artifacts
Why is simple staining not very useful for examining living organisms or to test an orgamism’s motility?
- because many of the dyes re toxic to the cells and can paralyze flagella, cilia and so forth
Living cells have a net (negative or positive) charge and negative staining uses (negatively or positively) charged dye which is repelled by the cell’s charge.
Negative, Negative
Why is it important NOT to heat-fix a smear that will undergo negative staining?
bc with negative staining you’re usually trying to identify heat-sensitive structures and heat fixation can distort the morphology and size of a cell
Why do physicians rely on gram staining results for prescribing antibiotics?
- some antibiotics will kill gram positive, some will kill/inhibit gram negative bacteria. Some will kill a wide array of bacteria including normal flora
Why do older cultures stain unreliably with gram stain protocol?
- waste products accumulate and break down cell walls as cultures age. Cells that usually stain gram positive will look gram negative
What genera of bacteria may not gram stain reliably?
- the mycobacterium species wont stain. They’ll look barely visible
How does iodine function?
iodine acts as a mordant. It forms a molecular bond with the crystal-violet
Gram negative cells have ________ cell walls surrounded by _________. They will _______ the crystal-violet-iodine complex during decolorization.
very thin, outter lipid membrane called lipopolysaccharide, not retain
Gram positive cells have ______________ cell walls. They will _______ the crystal-violet-iodine complex during decolorization.
very thick, retain
if a gram positive cell is overdecolorized, it will be the color _____.
red
if a gram negative cell is underdecolorized it will be the color ______.
purple
Skipping the iodine step means that gram positive cells will appear _________ at the end of the protocol.
red
What cell wall structure causes the stringing in the KOH test?
the DNA
Why is the gram stain result important for treating physicians?
- for prescribing an appropriate antibiotic and to learn the morphology and arrangement of the organism
Which test(s) yields the quickest result ? KOH, Vancomycin or gram stain
KOH #2 gram #3 vancomycian
What is the advantage of performing all 3 test (KOH Vancomycian and gram stain) on an unknown specimen?
- the three tests together provide a more accurate result
What are the two genera of endospore-forming bacteria mentioned in exercise 9?
bacillus spp.
clostridium spp.
Endospores are (metabolically active or inactive) while vegetative cells are (metabolically active or inactive) ?
inactive, active
What is the purpose of steaming the slide while staiing the Malachite Green?
it increases the porosity of spore coat so stain can penetrate
Define sporogenesis
the creation of an endospore
Define germination
the conversion of an endospore to a vegetative cell
What color will endospores and free spores be following the staining protocol?
green
What color will vegetative cells be following the staining protocol?
red
The role of water in the endospore staining protocol is (2)..
- to rinse the slide of excess stain, and to decolorize malachite green from vegetative cells
Why might a young (18 hour) culture of endospore-forming bacteria fail to have endospores? explain
because to produce an endospore the cell must be in harsh conditions. A young cell is not yet nutritionally stresses.
What are three functions that capsules serve?
- protect cell from desiccation
- protect from phagocytosis
- allows organisms to stick to each other
Water functions as a _____ in the capsule stain.
decolorizer
Is congo red a negative or positive stain? does it stain the organism or the background?
negative stain, stains the background
Is maneval’s a negative or positive stain? does it stain the background or the organism?
- positive stain, stains the organism
How does TTC function in motility media?
- as a tracking dye
What might cause false positive results in the test using motility medium?
you could have contaminated probe…it could have moved a bit inside the medium, gas bubbles could’ve exploded and shot organisms outward
Why is motility medium semi-solid?
it ensure that the motile organism will be able to swim away from the stab line
when performing hanging drop, how is it possible to distinguish true motility from brownian motion?
make sure they are moving in a purposeful direction not not just vibrating in place. make sure you’re not mistaking dehydration for movement
Describe proteus vulgaris flagellar arrangement.
- had flagella all around the edges, peritrichous
Describe spirillum voluntans flagellar arrangement.
- only had flagella on the opposite ends (amphirichous)
What was the purpose of flame sterilizing the inoculation loop in between quadrants during the streak plate technique?
- to ensure you don’t pick up too much bacteria into the next quadrant. flaming it ensures you start with a clean loop into the next quadrant.
If you have growth in quadrant one but none in 2-4 what may have caused this? streak plate technique
- not waiting for the inoculation loop to cool can kill the organisms not allowing it to grow
What sample from your own body could you have used as a mixed culture during the streak plate technique?
- nose, mouth, feces, sputum, saliva, sweat
Why is it so important to isolate organisms from a mixed culture into a pure culture?
for species identification, antibiotic susceptibility testing and other necessary tests
- can’t carry out copulates with a mixed culture
Define viable
only counts the LIVING cells
what is dilution?
- dilution is reducting the concentration of a substance.
- it’s a fraction and in scientific notation is a negative number
what is dilution factor?
- the FACTOR by which the substance has been diluted.
- the reciprocal of the dilution
How is MacConkey Agar both selective and differential?
- it is used in selective for detecting enteric bacteria (gram negative). It is used as a differential media when differentiating between lactose fermenters (pinkish colonies)and non-lactose fermenters (beige color)
How would you characterize a dilution series plate containing 419 CFUs?
too numerous to count (TNTC)
How would you characterize a dilution series plate containing 28 CFUs?
too few to count (TFTC)
How many CFUs/ml are generally present in the urine of young women?
10^2 - 10^3 mixed bacterial colonies
For the urine test, why might an uncircumcised male yield higher colony counts than an circumcised male?
the difference in anatomy
- mucous membranes cover urethra and bacteria is in this
How are quantitative and semi-quantitative counts different?
quantitative count are used to count the NUMBER of living bacteria in a unit volume
- semi-quantitative is a rough estimate in an unknown volume
What color are the colonies produced by E.coli on BBL chrome agar orientation medium
dark rose-pink
What color are the colonies produced by KES on BBL chrome agar orientation medium
medium blue to dark blue
What color are the colonies produced by PMP on BBL chrome agar orientation medium
pale-beige colonies surrounded by dark halos
Steps of gram stain
- Crystal violet
- Rinse with water
- Iodine (mordant)
- Rinse with water
- Decolorize with ethanol/acetone
- Rinse with water
- Counterstain with safranin
Steps of capsule stain
- Congo red to emulsify cells (negative stain that gives background color
- Maneval’s stain (positive stain to give cells color)
- Decolorize with water (to decolorize the capsule)
*smears are not heat-fixed
Steps of endospore staining?
- Steam smear and stain with Malachite Green (steam drives MG into spore coat)
- Decolorize vegetative cells with water
- Counterstain vegetative cells with Safranin
Antibiotics are ___ that word to inhibit or ____ microorganisms.
chemicals, kill
Antimicrobials that target bacteria are usually called _____
- antibiotics
An antimicrobial that kills bacteria
bactericidial
an antibiotic that inhibits bacteria
bacteriostatic
The antibiotic targets in bacterial cells
peptidoglycan, 70’s ribosome, bacterial nucleic acid synthesis enzymes, antimetabolites, cell membrane
Penicillin is ___ and inhibits ________
bactericidal, peptidoglycan synthsis
Synthetic sulfonamides are ___ and are considered antimetabolites
bacteriostatic
Quinolones interfere with _____replication
DNA
Tetracycline interferes with bacterial ______ by inhibiting the _______________ ribosome.
protein synthesis, 70s
Define broad spectrum
antibiotics that are effective against a wide array of bactria
Define narrow spectrum
antibiotics that are effective aainst a narrow array of bacteria
Define antibiotic
substance produced by one microorganism (typically a fungal or bacterial organism) that inhibits or kills another microorganism.
Define semisynthetic antibiotic
chemically altered version of a naturally-produced antibiotic.
Define zone of inhibition
the clear ring around an antimicrobial disk. It represents an area of growth inhibition.
Define Mueller-Hinton agar
Medium used; contains added buffers to prevent wide fluctuations in pH, that might otherwise alter the effectiveness of the antimicrobials being tested.
