Microbiology: Aeseptic Techniques

Front 1

PURPOSE of Aseptic Technique and Culture Handling

Back 1

To learn how to handle cultures in an aseptic manner.

To learn how to inoculate various types of media and to identify various types of growth.

Front 2

an ARTIFICIAL FORMULATION of nutrients and other essential substances required for microorganisms’ survival and reproduction

Back 2

Culture medium

Front 3

a culture medium that is LIQUID at room temperature

Back 3

Nutrient Broth (TSB)

Front 4

a medium containing PEPTONE and BEEF EXTRACT which is solidified with AGAR, a GELLING agent

Back 4

Nutrient Agar (slant) TSA

Front 5

a SPECIFIC MICROBE grown on a medium

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Pure culture

Front 6

the PROCESS by which microbes are TRANSFERRED from one medium to another using an inoculating instrument

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Subculturing

Front 7

Always incubate all bacterial plates in an _______ position.

This prevents ____________ from gathering on the ____ and dripping onto the ____ surface.

Back 7

inverted

condensation

lid

agar

Front 8

Please stack the _____ plates in the ________ to conserve space and be sure to put your plates on the appropriate shelf for your lab section.

Back 8

Petri

incubators

Front 9

When subculturing why must we use aseptic techniques?

Back 9

To insure the original stock and the new stock do not become contaminated.

Front 10

What is the first thing to do with the Petri plate?

Back 10

Label the bottom with a Sharpie.

Front 11

When pouring a Petri plate using molten agar in a tube, be sure to temper or cool the agar to _____ prior to pouring.

Back 11

~50 degrees

Front 12

When removing the cap drom the tube of agar what must you do with the cap?

Back 12

As soon as you remove the lid, discard the cap (This is the only time in Micro lab when it is OK to put test tube caps on your lab bench).

Front 13

What do you do with the lip of the tube?

Back 13

Flame it

Front 14

Why must you wipe off any water from the outside of the tube?

Back 14

This prevents contaminated water from dripping into your sterile Petri plate.

Front 15

Raise the lid of the petri plate, and pour the molten agar into the ________ of the dish while holding the Petri dish lid ______ the tube and plate bottom.

Back 15

bottom of the dish

above the tube

Front 16

Holding the Petri dish lid above the tube and plate bottom prevents what?

Back 16

contaminants from falling into the freshly poured agar

Front 17

Leave the Petri plate stationary until _________.

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solidified

Front 18

What happens if you stack the plates?

Back 18

It will take longer for them to cool.

Front 19

Once solidified, the agar will have what kind of appearance?

Back 19

An opaque appearance.

Front 20

What must you do before removing a small sample of bacteria from the available culture?

Back 20

Sterilize an inoculating loop

Front 21

What must you do before you begin to streak the Petri plate?.

Back 21

Recap or close the culture once you have removed your sample.

Front 22

Lift the lid of the Petri plate. Streak quadrant #1 so that the bacteria are evenly spread. Then put the lid back onto the agar. (How must the lid be placed on the bench?)

Back 22

Never put the lid on the bench so that the inside of the lid faces the bench. The inside of the lid will pick up bacteria from the bench.

Front 23

After the first streak and after lid is put back on what is the next step?

Back 23

Flame the loop.

Front 24

After flaming loop, Touch one corner of quad. #1 pulling a sample into what quadrant?

Back 24

#2 and spread evenly

Front 25

What do you do after spreading sample into quad. #2?

Back 25

Flame the loop again.

Front 26

Repeat steps of flaming ____and removing sample from _______quadrants.

Back 26

loop

previous

Front 27

You must flame the loop after each quadrant procedure ?

True or False?

Back 27

True

Front 28

The lid can stay off during each procedure?

True or False?

Back 28

False

Front 29

How can you est the coolness of your loop?

Back 29

You can test by touching the agar around the edge of the Petri plate. If it sizzles, its too hot. If it doesn't then it is ready.

Front 30

What is the goal of streaking a plate?

Back 30

To dilute out the number of bacteria you are moving with each additional streak.

Front 31

The division of individual cells

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Binary fission

Front 32

A population of clones that pile on top of each other and form this.

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Colony

Front 33

When you select a colony, you can be sure that you have generated a _____ ______.

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pure culture

Front 34

Why is the streak plate technique important when you have two unknown organisms?

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Because you want an isolated colony of each organism to make working stocks.

Front 35

What happens if you get a lot of moisture build up on nthe agar surface?

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You will not have colonies and you will generate a bacterial lawn.

Front 36

When oculating a slant, select bacteria using a ________ and use ___________.

Back 36

sterile loop

aeseptic technique

Front 37

Flame the lip of the sterile agar slant keeping the cap in the crook of your ______ ______.

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pinkie finger

Front 38

Why must you not put cap down on the bench?

Back 38

Because you will pick up contaminents.

Front 39

Why must you always check the agar and broth cultures you are oculating?

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To insure there are no contaminating organisms already growing.

Front 40

Where do you begin when inoculating a slant?

Back 40

Begin at the bottom of the slant

Front 41

Touch the agar surface and make a _________ pattern across the agar surface moving _____ toward the opening of the tube.

Back 41

squiggle pattern

moving up toward

Front 42

Be careful not to _______the agar surface.

When removing bacteria from an agar slant, do not ____ into the agar with the loop.

Back 42

do not to gouge

do not dig into

Front 43

How should you label the slants?

Back 43

Slants should be labeled with your name, lab section, date, instructor's name and organism name.

Front 44

When working with aerobic or facultatively anaerobic organisms, keep the cap _______ ________ during incubation.

Back 44

slightly loose

Front 45

When Inoculating a Deep, use an __________ ___________..

Back 45

Inoculating needle

Front 46

If you do not have an inoculating needle, you can _________________?

Back 46

you can straighten out a loop.

Front 47

Flame the needle and remove a small amount of ____________ ________.

Back 47

bacterial growth

Front 48

Stab the ________ through the agar or gelatin. Go straight ___ and straight _____.

Back 48

inoculum

straight in and straight out

Front 49

If your inoculating needle is too short, be careful not to stab the handle of the inoculating needle into the agar because ____?

Back 49

Because you have probably not sterilized the handle.

Front 50

After bacteria grow in an agar deep, you will see ______ along the _____ ______when you hold the tube up to the light.

Back 50

growth along the stab line

Front 51

If the organism is also _____ motile, you will see _____ ______ of the organism from the initial stab line throughout the agar.

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motile

outward motility

Front 52

Is the pattern wider or narrower at the top of the tube?

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Wider

Front 53

What does this pattern represent?

Back 53

Inverted Christmas tree

since most of our organisms are aerobes and fstest growth will occur toward the top of the tube.

Front 54

In gelatin, you will find that some bacteria are capable of breaking down protein in the gelatin resulting in ___________.

Back 54

Liquefaction

Front 55

Stabs should be labeled with what information?

Back 55

your name, lab section, date, instructor's name and organism name.

Front 56

When working with aerobic or facultatively anaerobic organisms, keep the cap ________ _________ during incubation.

Back 56

slightly loose

Front 57

When Inoculating a Liquid Broth, remove an inoculum of bacteria from a culture using a sterile ___________ ________.

Back 57

Inoculating loop

Front 58

What is the purpose of flaming the lip of the sterile broth culture and tilting the tube?

Back 58

so that the liquid can be reached with the inoculating loop w/o having to put the handle of the loop into the tube.

Front 59

What is the purpose of Twirling the loop around?

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so that bacteria are removed

Front 60

You will not be able to see the inoculum in the liquid unless_______?

Back 60

you have added too much.

Front 61

Another technique is to transfer the inoculum from ____ _____ to the _____ ______ of the test tube you are inoculating.

Back 61

from loop to the inside wall

Front 62

This technique will usually only work if ____?

Back 62

If the inside of the tube is not wet which means you should not shake or tilt the tube prior to this procedure.

Front 63

Once you have an inoculum stuck to the inside glass wall, you can tilt the tube so that _______?

Back 63

so that the bacteria are washed from the wall andf into the medium.

Front 64

What is the goal of this technique?

Back 64

To prevent introduction of the inoculating loop handle into the sterile tube.

Front 65

What is the purpose of heating the handle of an inoculating loop?

Back 65

to cut down on possible contamination

Front 66

Why must you not try to touch the handle of the loop against the mouth of the tube?

Back 66

This procedure will lead to contamination and spillage if the cap is not tight.

Front 67

Broth cultures should be labeled with what info?

Back 67

your name, lab section and organism name.

Front 68

An enteric organism; isolated from the intestines of animals.

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E. coli

Front 69

What are the colors of the colonies in E. coli?

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Beige and Mucoid

Front 70

Cells are very short bacilli with __________ _________.

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Peritrichous flagella

Front 71

Also enteric like the E. coli

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S. marcescens

Front 72

Colonies of S marcescens are what color?

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beige/pink at 37 degrees Celcius

Front 73

E.coli and S. marcesces are both ______________ pathogens.

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opportunistic

Front 74

organism found in soil and on skin of some animals

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M. leutus

Front 75

Is M. leutus a pathogen?

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NO

Front 76

Describe the colonies of M. leutus.

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Citron (sunshine) yellow
Have round margins and are raised (convex). Cells are cocci which occur in arrangements of tetrads. Non-motile.

Front 77

Premade, sterilized and stored on the shelves at the front of the lab.

Back 77

TSB, TSA, slants and motility

Front 78

How do you melt deep TSA plates?

Back 78

In boiling water baths at the back of the lab.

Front 79

Agar melts at _______Degree celcius.

Back 79

100 degree celcius (boiling point)

Front 80

Once melted the deeps are transferred to the _______ holding tanks where they will be ________.

Back 80

50 degrees celcius holding tanks


cooled

Front 81

Agar will solidify at what temperature?

Back 81

45 degrees Celcius

Front 82

TSB is inoculated with an ______ _______ and incubated at what degrees for E.coli and what degrees for M. luteus?

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inoculating loop

37 degrees celcius and 25 degree celcius

Front 83

TSA plates are inoculated using ______ ________streak plate technique and incubated in an _________ position at temperatures as indicated above.

Back 83

four quadrant technique

inverted

Front 84

Motility agar is _______ using an __________ needle and incubated with loose caps.

Back 84

stabbed

inoculating needle

Front 85

TSA slants are ____________by ___________ the slant form bottom to top with bacteria usin and ________ ________.

Back 85

inoculated

streaking

inoculating loop

Front 86

Slants are incubated at what temperature?

Back 86

37 degrees celcius

Front 87

a general purpose liquid medium used to grow cultures

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TSB

Front 88

another word for turbid

Back 88

cloudy

Front 89

the ability of bacteria to move
(flagella)

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motility

Front 90

Relatively clear and allows you to visualize movement of bacteria through the agar in all directionsaway from the stab line.

Back 90

Motility agar

Front 91

smooth, complete coverage of the slant w/o contamination

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TSA Slants

Front 92

These slanta are perfect for storing large numvers of organisms on a relatively large surface that is sealable with a lid.

Back 92

TSA slants

Front 93

Cultures may be preserved for long periods of time in the refrigerator on ________.

Back 93

slants

Front 94

What produces a red pigment giving teh broth a red appearance?

Back 94

S. marcescens

Front 95

Usually are of a different color or texture and often do not lie on the streak line.

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Contaminants

Front 96

M. Luteus is motile or non-motile?

Back 96

nonmotile

Front 97

E.coli and S. marcescens are non motile or motile.

Back 97

motile.

Front 98

Wide Ribbon-like pattern of growth appears if _____?

Back 98

If you shake when inoculating the agar.