Microbiology Lab Practical 1 Flashcards by manda hare (2022)

Table of Contents
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) 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.

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

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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?

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?

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?

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

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