Microbiology Lab Test #1

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sepsis

Back 1

growth of decay, putrification, infection in body & blood tissues

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asepsis

Back 2

any procedure that prevents infections

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aseptic technique

Back 3

practices-physical or chemical- that exclude all organisms from contaminating media & prevent from return

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sterilization

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a process that destroys all living microorganisms, non-living viruses, endospores by steam (autoclave), dry heat, incineration, chemicals or radiation (UV box); absolute value when all infections are destroyed

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disinfection

Back 5

a process where vegetative non-endospores are destroyed on non-living objects or surfaces

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antiseptics

Back 6

chemical agents that are used on human tissue to destroy or inhibit vegetative bacteria

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bacteria, a microorganism

Back 7

organisms that are huge in numbers, widely distributed everywhere it's best adapted; are not always confined to those areas; transfers easily to other environments; special procedures impt. (aseptic technique) to handle them

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media

Back 8

any nutrient substance that can support microbial growth

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culture

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microorganisms growing in or on media

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inoculation

Back 10

procedure where a microorganism is introduced to a sterile media

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fomite

Back 11

any non-living object that can carry bacteria

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ultrastructure

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the fine details of organisms, tissues and cells.

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magnification

Back 13

the ability of lenses to make an image larger

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resolution

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the ability of lenses to see the fine details of an image in clear, sharp, focus

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ocular lens (eyepiece)

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the 10X lens in a compound microscope that magnifies and resolves the image made by the objective lens

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4X

Back 16

objective lens:____
color: red ring
magnification: 40X
power: scanning power

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10x

Back 17

objective lens: _____
color: yellow ring
magnification: 100X
power: low power

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40X

Back 18

objective lens: ____
color: blue ring
magnification: 400X
power: high power

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100X

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objective lens:___
color: white ring
magnification: 1000X
power: oil immersion power

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objective lens

Back 20

the lens in a compound microscope that does the initial magnification of the specimen on the microscope slide; things that rotate

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morphology of bacteria

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cell size, shape, arrangement

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monomorphic

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rigid cell wall w/ peptidoglycan; one permanent shape

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describe ultrastructure of bacteria

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bacteria are small, colorless and transparent; needs to be stained to be seen and identified

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stain

Back 24

a color dye used to show specific cells on a smear when viewing in a microscope

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negative stain

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a staining procedure that results in colorless, clear bacteria against dark background; visible clear cell morphology against dark field

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chromophore

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the color-bearing ion in a stain; if chromophore is anionic (-), repelled by bacteria which has 20% (-) nucleic acid charge

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nigrosine

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aka india ink are used for negative staining (background stain)

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heat fixing

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organisms are allowed to dry on a glass microscope slide then passed through quickly & briefly on flame to kill & fix cells to slide, melt cell walls to stain better.

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heat fixing v.s. background staining

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* heat fixing doesn't allow bacteria to be seen in natural state

* ruptures & distorts cells

*negative stain is most accurate to determine cell morphology

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what a good heat-fixed smear should be

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1) must be able to withstand one or more washings w/o loss of microorganisms

2) does not distort cell excessively

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heat fixing: what could go wrong? (7)

1) too much heat: cells won't stain & won't be seen under microscope

2) too little heat: cells are not killed and won't stain well

3) if not completely air dried before heat fixing, cells boil, rupture and won't stick to slide

Back 31

4) too thick: light can't pass through, can't see individual cells

5) too thin: not enough cells to see

6) if too few cells from liquid broth: use 3-4 loopfuls

7) if too thick from solid media: too many cell colonies-dilute 1 loopful of culture with 1-2 loopfuls of distilled water=1:2

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simple stain

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1) a single dye added to smear
2) a one-step procedure
3) the use of a single stain to
color a cell

- methylene blue is used as simple stain

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basic stain

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the chromophore is cationic (+); will stain slightly (-) bacteria
- used to find out cell morphology

-methylene blue is a basic stain

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rule on simple stain & basic stain

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rule on simple stain & basic stain:

bacterial cell walls are negatively charged and are stained by basic stains

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Christian Gram

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Danish bacteriologist who developed a staining technique that separates bacteria into 2 groups: G+; G-

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G+ bacteria

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* microorganisms really thick cell walls

* are able to retain the PURPLE color of crystal violet iodine & resist decoloration with ETOH

ex. cocci, chains of bacilli

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G- bacteria

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* decolorized by ETOH by removing outer lipid layer from its cell walls
* counter-stained with safranin
* red or pink color

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differential stain

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a stain that distinguishes bacteria based on their reaction to the staining procedure due to their structural differences

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mordant

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1) a chemical substance added to a staining solution to make it stain more intensely; it is NOT a stain but an enhancer

2) a fixative which bonds chemically with CV-I

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What happens in Gram staining?

* the cell wall accepts the primary stain (Crystal violet) that reacts with the Gram's iodine (mordant=CV-I)) to form an insoluble precipitate (CV-I bond)

* the G- bacteria's outer lipid layer is washed off by decolorizer (ETOH), increases pore size of cell walls and removes CV-I & becomes colorless

Back 40

(note: G- bacteria is resistant to lysozomes and antibiotics and are more virulent than G+ bacteria)

*procedure:
1. primary stain=60 secs. (crystal violet)
2. mordant - 60 secs. (Gram's iodine) (CV-I)
3. decolorizer - 20 secs. (ETOH)
4. counterstain - 60 secs. (safranin)

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What can go wrong in Gram staining? (5)

1) too thick, the smear will not decolorize

2) age - old cultures tend to be Gram variable; over 24-hrs may contain non-viable cells that begin to decompose = cells decolorize causing G+ cells to apper as G-.

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3) overheating makes cells burst and overdecolorize

4) overuse of decolorizer

5) underuse of decolorizer

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culture

Back 42

microorganisms that grow and multiply in or on solid or liquid medium

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culture medium

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any nutrient substance that can support microbial growth

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mixed culture

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* mixed bacteria growing together on a culture medium

* commonly found in nature, on body, soil, water and food

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pure culture

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* only one specific microorganism growing on a culture medium

* rare in nature, occurs under extreme environmental conditions

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isolation

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the process of separating an individual microorganism from a mixed population = to produce pure culture

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Why is it necessary to have a pure culture? (5)

1) the Germ Theory (Koch's Postulates) specifically calls for a pure culture

2) to identify unknown bacteria

3) to identify specific spoilage and pathogenic microorganisms

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4) Pasteur used this to explain fermentation (why wine sours)

5) to test for quality & safety of food, milk and water

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Facts that make pure culture possible:

as bacteria grow and multiply on the surface of solid media, they form clumps or colonies

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* a single bacterial cell yields colony

* colony morphology

* pure colony is made by aseptic transfer of inoculum to fresh medium via streak plate method or pour plate method

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colony

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a visible mass of bacterial cells on the surface of solid media

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inoculum

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the microorganisms introduced to a sterile medium

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dental caries

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* breakdown of tooth enamel and dentin to form tooth decay

*produces lactic acid w/ high levels of sucrose

* bacterial infection and a worldwide widespread chronic disease

* microbes in saliva
(pH 7) and tooth plaque produce acid

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streptococcus mutans

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is the microorganism found on surface of decayed teeth

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lactobacillus

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bacterial species most commonly involved in tooth decay (not on it)

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similarities between streptoccocus mutans and lactobacillus

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are microorganisms which produce acid and have high tolerance for acid in mouth

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Snyder Test Agar

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* this caries test is simple and highly reliable

*relies on the rapidity of the bacteria in saliva to lower the pH in a medium that contains 2% dextrose

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decalcification

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* this signals breakdown of enamel at pH 5.5 (strong) and lowers to pH 4.4 (stronger) and less

*pH lowering is evidence of susceptibility to dental caries

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What Snyder Test Agar contain?

Back 57

2% dextrose
bromcresol green (pH indicator)

*green down to pH 4.8 becomes yellow at pH 4.4 and below agar

Results:
Green = (negative)
Yellow = (positive) for tooth decay

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transient

Back 58

contaminants that are not permanent skin residents, easy to remove and transmitted by fomites

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fomites

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any non-living inanimate object that can carry and transmit bacteria

* only on skin for few hours or few days

* sensitive to antiseptics (alcohol pad)

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resident microorganisms

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* are normal microbiota entrenched in and on the human body which do not cause infection under
normal conditions

* slowly removed by handwashing

* low pathogenic Staph species

* less sensitive to antiseptics

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degermers

Back 61

* agents that physically remove from a limited area of the skin

* affects vegetative pathogens

* best kind is alcohol pad

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selective media

Back 62

* a media that contains chemicals that prevent growth of unwanted bacteria

* used to isolate specific bacteria from mixed culture

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differential media

Back 63

* a media that indicates the presence of a specific bacteria usually by color change due to bacteria's unique metabolic step

* a color change in the media indicates a positive reaction following inoculation and incubation

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Mannitol Salt Agar (MSA)

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is a selective and differential media

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What's in MSA plate?

1) mannitol - sugar that only Staph A can ferment to produce acid (differential)

2) has 7.5 NaCl - salt concentration that inhibits other bacteria except salt-tolerant Staph A (selective)

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3) phenol red - a pH indicator; range of 6.8-8.4 pH
- basic (above pH 7) = red
- neutral (pH 7) = red
- acid (below pH 7) = yellow

* mixed bacteria on MSA but only Staph A present due to salt

* yellow means acid formed by Staph a

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Streptococcus pyogenes

Back 66

* medically important species of Streptococci

* responsible for more illnesses and cause greater variety of diseases than any other bacteria group

* causes beta-hemolysis

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beta-hemolysis

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complete destruction of RBCs on a blood agar plate = clear zone

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alpha-hemolysis

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incomplete destruction of RBCs on blood agar plate = brownish green