Lab Practical

Front 1

Diplococci

Back 1

pairs that divide in one plane

Front 2

streptococci

Back 2

chains that divide in one plane

Front 3

tetracocci

Back 3

tetrads that divide in two planes

Front 4

staphylococci

Back 4

clusters that divide in three planes irregularly

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sarcinae

Back 5

cuboidal packets that divide in three planes regularly

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coccobacilli

Back 6

cocci that are slightly longer in one direction

Front 7

fusiform bacilli

Back 7

tapered at the ends, appearing football like

Front 8

filamentous bacillary

Back 8

long threads

Front 9

helicoid

Back 9

grow in spirals

Front 10

Cationic dyes

Back 10

positively charged chromophore

basic

methylene blue and crystal violet

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Anionic dyes

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negatively charged chromophore

acidic

Nigrosin, acid fuschin, Congo red

Front 12

Fat Soluble Dyes

Back 12

no charge

Sudan Black stains granules of poly-B-OH-butyric

Front 13

Insoluble dyes

Back 13

water insoluble

India ink, colloid suspension of carbon particles

Front 14

Negative Staining

Back 14

no heat fixing

good for morphology

stains background

can use Nigrosin or India Ink

Front 15

Simple Staining

Back 15

stains all bacteria cells

cationic dyes are used because they attract the negative cell surface

methylene blue or crystal violet

Front 16

Acid Fast Stain

Back 16

differentiates based on cell wall characteristics

acid fast - high wax content

Cells are steamed with carbol fuschin

decolorized with acid alcohol

counterstained with methylene blue

Acid fast will remain red

others will pick up the blue dye

Front 17

what is another name for the acid fast stain?

Back 17

Ziehl Neelson

Front 18

Two types of acid fast microorganisms

Back 18

Mycobacterium

Nocardia: less harsh decolorizer must be used

Front 19

Gram stain

Back 19

stains based on cell wall peptidoglycan

Crystal Violet

Grams Iodine

Ethanol

Safranin

positive is purple

negative is pink

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Spore Stain

Back 20

also called the Schaeffer Fulton method

Steamed with malachite green

decolorized with water

counterstained with safranin

endospores and freespores - green

vegetative cells - pink

Front 21

Pour

Back 21

15-20 ml of liquid agar in a pour plate

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broth

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5-7 ml of liquid media

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deep

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5-7 ml solidified upright

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Slant

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5-7 ml of media solidified at an angle

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fermentation broth

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broth with durham tube added

Front 26

Natural media

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unknown composition

example is nutrient agar

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synthetic media

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known composition

designed for selection and differentiation

Front 28

PEA

Back 28

phenylethyl alcohol agar

selects for gram + organisms

Front 29

DES

Back 29

desoxycholate agar

selects for gram - organisms

it also differentiates lactose fermenters

when lactose fermenters produce acid they precipitate bile salts and absorb the natural red dye

red = + for lactose fermentation

Front 30

EMB

Back 30

Eosin Methylene Blue

selects for gram -

differentiates lactose fermenters

Lactose + will show a color change

Front 31

Further differentiation on EMB

Back 31

Mixed Acid: produce more acid and have dark blue black centers. E coli has green sheen.

2,3 butanediol: produce less acid, colonies have pale pink to lavender centers. bulls eye colonies.

enterobacter example.

Front 32

Blood Agar

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Differentiates presence or absence of hemolysins.

B: complete hemolysis

A: partial hemolysis, sometimes green bc billiverdin

G: no hemolysis

Front 33

Starch Agar

Back 33

tests for presence of amylase which breaks down starch.

Iodine is added to the plate.

If blue appears, the result is negative and amylase is not present.

Front 34

Milk Agar

Back 34

tests for the presence of caseinase which hydrolyzes casein in to amino acids.

since casein gives milk its white color the presence of caseinase would cause clearing.

clearing = + for caseinase

Front 35

Lipase Plate

Back 35

tests for lipase which breaks down fat to form glycerol and fatty acids.

Indicator is SPIRIT BLUE DYE.

fatty acids lower the ph enough to produce a dark blue precip when lipase +.

starts blue and intensifies.

Front 36

Sugar Fermentation Tubes

Back 36

used to see if an organism can ferment either glucose, lactose, or mannitol.

has the sugar, indicator phenol red, and durham tube.

yellow = acid, fermented

red = negative

yellow and gas = fermented acid and gas

cerise = alkaline

Front 37

Methyl Red (MR)

Back 37

tests for mixed acid fermenters.

acid lowers the ph below 5.1 so when methyl red is added to the culture it remains red.

Red is a positive result.

Ex. Escherichia

Front 38

Voges-Proskauer (VP)

Back 38

tests for 2,3 butanediol fermenters which produce less acid.

Since AMC is easier to detect you look for that.

Add Barritts reagents.

Brick red color indicates is a 2,3 butandiol fermenter and is a positive result.

ex. Enterobacter

Front 39

What are Barritt's Reagents?

Back 39

1. Add VP1 (a napthol) and VP2 (KOH)

2. When o2 is present VP2 reacts with AMC and produces a brick red color

3. VP1 is used to intensify the color

Enterobacter is VP +

Front 40

Catalase Test

Back 40

tests for the presence of catalase which converts H2O2 to water and oxygen.

this formula is used to eliminate toxic H2O2.

1. Add 3% H2O2 to the culture.

2. Bubbles = + for catalase

Front 41

Oxidase Test

Back 41

tests for the presence of cytochrome c oxidase.

this is used to oxidize aromatic amines and form a colored product.

1. di methyl phenyldiamine hydrochloride will turn a dark blue black color in the presence of oxidase.

Front 42

Nitrate Test

Back 42

tests for nitrate reductase.

1. add n1 and n2 to the tube.

n1=sulfanilic acid n2=di methyl a napthylamine

2. if brick red color appears result is +.

3. Add zinc

4. If red color appears, the result is -.

5. if the tube stays clear, this is a + result which indicates the presence of both nitrate and nitrite reductase.

Front 43

Tryptone (Indole)

Back 43

tests for tryptophanase which converts tryptophan in to indole and pyruvic acid.

1. You can test for indole by adding Kovac's reagents.

2. Red ring color is a + result.

Kovac's: DMABA (dimethylaminobenzaldehyde) and HCl dissolved in amyl or butyl alcohol.

Front 44

Urea Test

Back 44

tests for the enzyme urease which converts urea in to ammonia and CO2.

Substrate is urea and indicator is phenol red.

When ammonia is released the ph increases above 8.1 and a cerise appears.

This is a + result.

Example is Proteus.

Front 45

Hydrogen Sulfide Production (H2S)

Back 45

tests for cysteine desulfurase which removes the sulfur side chain from cysteine to produce H2S.

H2S in the presence of iron salts forms a black precipitate.

Black color = + for enzyme

Front 46

SIM Media

Back 46

tests for Sulfur reduction, Indole, and Motility.

H2S + = black precipitate.

Indole + = red after Kovaks is added.

Motility + = growth away from innoculation.

Front 47

Simmons Citrate

Back 47

test to see if an organism can use citrate as the sole carbon source.

media has ammonia, citrate, and bromothymol blue as the indicator.

+ Result = deep prussian blue growth

Front 48

Phenylalanine (PPA)

Back 48

tests for phenylalanase which turns phenylalanine in to PPA and NH3.

1. Add Ferric Chloride to the media which detects the presence of PPA.

2. + result is a deep green color.

Front 49

Litmus Milk

Back 49

Litmus milk has the ph indicator litmus and powdered milk. starts off purple.

1. Acid Rxn: pink liquid bc lactose fermentation

2. Acid Curd Rxn: pink solid. acid production bc Lac fermentation and coagulation of proteins.

3. Reduction: litmus becomes white.

4. Alkaline Rxn: tube becomes blue when ammonia increases the ph.

5. Peptonization/Proteolysis: clearing of the medium due to caseinase. can be brown or amber.

Front 50

Gelatin Test:

Back 50

tests for presence of gelatinase which hydrolyzes gelatin in to amino acids.

1. Stab inoculate tube.

2. Place on ice.

3. If solidifies it is - for gelatinase.

Front 51

IMViC

Back 51

differentiates between E Coli and EA.

Indol, MR, VP, Citrate

EC + + - -

EA - - + +

Front 52

Motility Media

Back 52

tests if bacteria are motile or not.

indicator is tetrazolium chloride.

red means growth.

growth away from the inoculation line indicates motility.

Front 53

Russel's Double Sugar (RDS)

Back 53

tests for fermentation of glucose and lactose as well as gas production.

indicator is phenol red.

media starts red and turns yellow post ferm.

yellow butt = glucose fermented

yellow slant = lactose fermented

read slant over butt

1% lactose and 0.1% glucose

24-48 hr read time

Front 54

Kligler's Iron Agar

Back 54

tests for ferm. of glucose and lactose, H2S, and gas production.

1% lactose and 0.1% glucose and iron salts

same fermentation results as RDS

Black precip = + H2S

Cracks or lifting = + gas

18 hr read time

Front 55

KIA bacteria examples

Back 55

PA: G- L- H2S-

Shigella: G+ L- H2S+

E Entero: G+ L+ H2S-

Salmonella Proteus: G+ L- H2S+

Front 56

OF Glucose Test

Back 56

Positive test starts purple.

Negative test starts green.

1. Two tubes are inoculated and one is covered in oil.

2. indicator is bromothymol blue

Both yellow tubes: strict fermentation

Both uninoculated color: strictly oxidative

Open yellow, Closed uninoculated: partial oxidative

Open uninoculated, closed yellow: faculative

Front 57

Bismuth Sulfide Agar (BSA)

Back 57

starts off dull green.

Ferrous sulfite is the H2S indicator.

Brilliant green dye and bismuth sulfite inhibit the growth of gram + and coliforms.

example: salmonella typhi produces black color

Front 58

Brilliant Green Agar (BGA)

Back 58

differentiates lactose and sucrose fermentation.

brilliant green dye inhibits gram + and coliforms.

phenol red is the indicator of lactose ferm.

lactose/sucrose ferm = yellow and green colonies

non lactose/sucrose ferm = opaque pink/red/white and surrounding media red

Front 59

SS Agar (light pink plate)

Back 59

contains bile salts, sodium citrate, and brilliant green to inhibit gram + and coliforms.

limits swarming by proteus.

natural red dye is an indicator of fermentation and + colonies appear red.

detects H2S.

Salmonella appears black.

Shigella appears colorless.

Front 60

Desoxycholate Citrate (light pink)

Back 60

selects for gram -

differentiates lactose fermenters

neutral red dye indicates acid production via fermentation.

+ colonies will appear red

Front 61

Coagulase

Back 61

tests for the enzyme coagulase which converts fibrinogen in to fibrin.

inoculate in a tube of rabbit plasma overnight.

clumpy = coagulase +.

only valid on gram + staph like bacteria.

Front 62

Phenol Red Mannitol Salt Agar

Back 62

only grows staph bc of high concentration of salt. 7.5%.

medium starts red but turns yellow if organism is mannitol positive.

Front 63

Staph 110 Medium

Back 63

same as PRMSA but it lacks the phenol red.

selects for staph but allows for growth of natural colony pigment.

Front 64

DNase

Back 64

tests for DNase which hydrolyzes DNA in to free nucleotides.

turquiose blue agar.

methyl green forms a complex with DNA in the media and if DNase is present it breaks this down and forms clearing.

clearing = + result

Front 65

m-Staph broth

Back 65

media containing 10% NaCl which selects for staph because of the high salt concentration.

Front 66

Endo Agar

Back 66

selects for gram -.

contains sodium sulfite and basic fuschin.

differentiates lactose fermenters.

lactose + = red colonies

coliforms produce golden metallic golden sheen.

Front 67

Cardinal temps of psychropiles

Back 67

0 - 5 - 15 to 20

Front 68

cardinal temps of mesophiles

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20 - 30 to 37 - 45

Front 69

cardinal temps of thermophiles

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45 - 55 to 65 - 70 to 90

Front 70

UVr

Back 70

ultraviolet resistant

Front 71

UVs

Back 71

ultraviolet sensitive and lethal