Microbiology Practical 2!!!! i will miss this class! (Ilana Kovach)

Microbiology 2420Practical 2

Ilana Kovach

Identification of Bacillus Species

Starch media Substrate:Polysaccharide Starch

Enzyme:Amylase & Maltase

starch

+

+

-

-Maltose (disaccharide)

Glucose (monosaccharide)

Amylase Maltase

Lipid mediaSubstrate:Tributyrin

Enzyme:Lipase

Triglycerides

+

+ (Slightly)

+

-

Glycerol

Fatty acids

Caesin MediaSubstrate:Caesin

Enzyme:Protease & Casease

Casein

++

+ -

Peptones Polypeptides dipeptidesAmino

acids

+

++ - -

-

Gelatin mediaSubstrate:Gelatin (Protein produced by hydrolysis of Collagen)

Enzyme:Gelatinase

Gelatin

+ -

Amino acid components “cause gelatin to liquefy”

Gelatinase

Mannitol Salt agarSelective (7.5% NaCl) halotolerant

Differential (Mannitol)

pH indicator:

phenol Red Red Yellow

Nitrate ReductionDifferential Only (Beef extract, Peptones & Nitrate)

Enzyme: Nitrate Reductase Anaerobically reduce nitrate nitrite Ammonia or completely to Nitrogen

(5 drops of Each)Nitrate A = Sulfanilic Acid

Nitrate B = Alpha-naphythylamine

Yellow Positive

(nitrates reduced beyond

nitrites to ammonia or

molecular nitrogen)

RED Positive

reduced Nitrate NitriteYellow Add Zinc

RED Negative

(The zinc reduced

the nitrate NOT the

bacteria)

Citrate UtilizationSelective Only (Sodium Citrate, Sodium & Water)

Enzyme: Citrase

pH indicator: Bromothymol Blue

CO2

Sodium

citrate

Oxaloactetic

acid

Acetic

acid

pyruvate

Alkaline

Sodium

bicarbonate

pH

Indicator

Reacts

with

Na+ & H2O

Reacts

with

Bile Esculin (BEA)Selective (Bile Salts)… Gram Negative MacConkey

Differential (Esculin & Ferric ammonia Citrate)Hydrolyze the carbohydrate Esculin

Esculin

EsculetinGlucose

Ferric Ammonia Citrate “form black precipitate”

Reacts

with

Transformation

Escherichia coli + pGreen Plasmid Fluorescent Escherichia Coli

*CaCl (increase permeability)

Competent Cells

Artificially competent

Vector

Plasmid vectors engineered to contain

1) Selectable marker

2) Cloning site

Transformation Efficacy# 𝒐𝒇 𝒕𝒓𝒂𝒏𝒔𝒇𝒐𝒓𝒎𝒂𝒕𝒂𝒏𝒕𝒔

𝒂𝒎𝒐𝒖𝒏𝒕 𝒐𝒇 𝑫𝑵𝑨 (𝒎𝒊𝒄𝒓𝒐𝒈𝒓𝒂𝒎𝒔)

TransformationLB Before: Growth

LB after: Growth

LBamp Before: No growth

“NOT resistant to ampicillin”

LBamp After: Growth

“Resistant to ampicillin” &

Glows

identification of StaphylococcusSpecies

Catalase TestH2O2 (Hydrogen Peroxide) 2H20 (Water) + O2 (Oxygen)

Staphylococcus +

Streptococcus/

Enterococcus -

Mannitol Salt Agar Selective (7.5% NaCl) halotolerant

Differential (Mannitol)

pH indicator:

phenol Red Red Yellow

+

+weak

+

-

DNase agar

Differential: Methyl green dye &

polymerized DNA form complex

which give the agar a Blue-Green

Color at pH 7.5

DNA

smaller

fragments

unbound to

methyl green resulting in a zone of clearing

+ +

- -

Mueller Hinton Agar Test Susceptibility of Novobiocin

>18mm

Nutrient agar (Neither

selective or differential) Controlled: Levels of thymine, Thymidine, Ca+ ions & Mg

+

+-

-

Rapid Staph TestLatex particles coated with human fibrinogen & IgG

Staphylococcus aureus

Particles will bind to any staph bacteria that

Contain coagulase/Protein A

“Protein A bind to Fc region of IgG antibodies”

resulting in visible

clumping or agglutination

of latex particles

Coagulase TestFibrin Fibrinogen Tube Test: inoculate into tube of citrated plasma

Staphylococcus Aureus +

Other staphylococcus Strands -

Identification of Streptococcus & enterococcus Species

Blood Agar & Antibiotic Susceptibility

Antibiotics: Bacitracin & SXT

Differential Media

Exotoxin (Hemolysis)

Beta Beta

AlphaAlpha

Bile Esculin (BEA)Selective (Bile Salts)… Gram Negative MacConkey

Differential (Esculin & Ferric ammonia Citrate)Hydrolyze the carbohydrate Esculin

Esculin

EsculetinGlucose

Ferric Ammonia Citrate “form black precipitate”

Reacts

with

Esculin

(+) hydrolyzed to glucose & esuletin

Mannitol Salt Agar Selective (7.5% NaCl) halotolerant….

Enterococcus + & Streptococcus -

Differential (Mannitol)

pH indicator:

phenol Red Red Yellow (Enterococcus Faecalis)

Rapid Strep TestSand which immunoassay: Test cassette contains a strip coated with

strep A antibodies

Strep A antigen

extracted from

throat swab

will bind to the

antibodies

the antigen antibody complex

then binds to a second antibody

that is conjugated with a

chromogen “colored line”

* Wrong 15% of the time

Identification of Gram Negative

MacConkey Agar Selective: Crystal Violet

& Bile salts *Inhibit Gram positive &

Select Gram Negative

Differential:Ferment Lactose?

1) fermented lactose lower pH of media

below 6.8 pinkish-Red

2) Did not ferment lactose “colorless”

CarbohydrateSubstrate Lactose

pH indicator Neutral Red

Citrate UtilizationSelective Only (Sodium Citrate, Sodium & Water)

Enzyme: Citrase

pH indicator: Bromothymol Blue

CO2

Sodium

citrate

Oxaloactetic

acid

Acetic

acid

pyruvate

Alkaline

Sodium

bicarbonate

pH

Indicator

Reacts

with

Na+ & H2O

Reacts

with

Urease Test

Enzyme:Urease

Substrate:Urea

pH Indicator Phenol Red

urea

ammonia carbon dioxide

& water

Phenol

red

Reacts

with

Nitrate ReductionDifferential Only (Beef extract, Peptones & Nitrate)

Enzyme: Nitrate Reductase Anaerobically reduce nitrate nitrite Ammonia or completely to Nitrogen

(5 drops of Each)Nitrate A = Sulfanilic Acid

Nitrate B = Alpha-naphythylamine

Yellow Positive

(nitrates reduced beyond

nitrites to ammonia or

molecular nitrogen)

RED Positive

reduced Nitrate NitriteYellow Add Zinc

RED Negative

(The zinc reduced

the nitrate NOT the

bacteria)

MRVP Test

Methyl Red (MR) Test Vogues- Proskaur (VP) Test

Ferment glucose & produce more than

one acid end product Lactic, Acidic

and formic acids

STRONG enough to overcome

phosphate buffering system

Positive

Reaction:Red pH of 4.4 or lower

Ferment glucose but only produce ONE acid

end product “usually acetic acid”

The acid produced initially lowers the pH of

the media but is quickly converted to

acetylmethylcarbinol which leads to a pH of

6.2. Reagents react with acetylmethycarbinol

Positive

Reaction:Red Band at the top which will diffuse

over time

Never can be Both!!!!!

*Can be both Negative

SIM agar Test Contains: Amino acid Tryptophan

(peptones, sodium thiosulfate & ferrous sulfate)

Indole Production: Tryptophanase degrade tryptophan into indole, pyruvate and ammonia.

*kovac’s Reagent contains HCL, butanol, and para-dimethyl-

amino-benzaldehyde (pdaba).

Hydrogen Sulfide Production Thiosulfate reductase can reduce sulfur to hydrogen sulfide gas

Motility: Motile vs. Nonmotile

“Sodium

thiosulfate”

Hydrogen sulfate

Ferrous sulfate

Reacts

with

Tryptophan

Indole

Pyruvate ammonia

acidified butanol

extracts any indole that is

produced & bring to

surface

pdaba

Reacts

with

Lysine Iron AgarDeamination Aerobic (slant)

Deaminate positive

Deaminate Negative

Decarboxylation Anaerobic (Butt)

Decarboxylation Positive

Decarboxylation Negative

Hydrogen SulfideThiosulfate reductase can reduce sulfur to hydrogen sulfide gas

0.1% Lysine, Glucose, Peptones, Bromcresol purple, Sodium thiosulfate, ferric ammonia citrate

ammonia ferric ammonium citrate α-ketocarboxylic acid

Reacts

with

Cadaverine “diamine end”Raise ph above 6.8

Bromcresol purpleCleaves carboxyl group from lysine

Reacts

with

Triple sugar Iron (TSI) Agar Test 0.1% glucose, 1.0% sucrose , 1.0% lactose, Peptones, Phenol red, Sodium thiosulfate, Ferrous sulfate

Carbohydrates Substrates (Slant & Butt)0.1% glucose, 1.0% sucrose, 1.0% lactose

*Fermentation of these carbohydrates into acid end products will result in

a yellow color (A) on the slant and/or Butt as the acid reacts with the

Phenol Red

*No fermentation results in a pink color (Alk)

GasPositive or Negative “look for cracks”

Hydrogen SulfideThiosulfate reductase can reduce sulfur to hydrogen sulfide gas

Organism Catalase MSA

fermentation

DNase Novobiocin

Sensitivity

Staphylococcus aureus + + + S

Staphylococcus epidermidis + - - S

Staphylococcus saprophyticus + + - R

Staphylococcus Xylosus + +/-

Weak

+ R

Organism Catalase Hemolysis Bile

Esculin

SXT Bacitracin MSA

growth

Enterococcus

Faecalis- α + S R +

Streptococcus

Bovis- α + R R -

Streptococcus

mutans- ϒ + S S -

Streptococcus

pyogenes- β - R S -

Streptococcus

zooepidemicus- β - S*

(usually 21mm

Barely)

R -

Triple Sugar Agar SIM deep Lysine Iron Agar

Gram Negative

Bacterium

Lact

ati

on

Fer

men

tati

on

Sla

nt

Bu

tt

Gas

H2S

Ure

ase

Cit

rate

Ind

ole

H2S

Moti

lity

*

MR

VP

Nit

rate

Sla

nt

Lysi

ne

dea

min

ati

on

Bu

tt L

ysi

ne

Dec

arb

oxyla

ti

on

H2S

*

Aeromonas

Hydrophila

- Alk A - - - + + - - + + - -

Alcaligenes

faecalis

- Alk Alk - - - + - - - - - - +

Citrobacter

freundii

+ A A + + - + - + + - + - -

Edwardsiella tarda - Alk A + + - - + + + - + - +

Enterobacter

aerogenes

+ A A + - - + - - - + + - +

Enterobacter

Cloacae

+ A A + - - + - - - + + - -

Escherichia coli + A A + - - - + - + - + - -

Morganella

morganii

- Alk A + - + + + Weak +

+ - + - -

Proteus mirabilis - Alk A - + + + - + + - + + -

Proteus vulgaris - A A - + + + + + + - + - -

Providencia

alcalifaciens

- Alk A - - - + - - + - + + -

Providencia

rettgeri

- Alk A - - + + + - + - + + -

Appendix A Day 1Gram + (Coccus)Gram – (Rod)

Nutrient Agar Plate Not selective

Not differential

C-CNA Agar PlateSelective: Colistin + Nalidixic Acid

Gram Positive

Differential: Blood agar

Beta, Alpha, Gamma

MacConkey Agar Selective: Crystal Violet & Bile Salts

Gram Negative

Differential: Substrate Lactose & pH

indicator “Neutral Red”

RED Pink

Gram Stain

Appendix A Day 2

Alcaligenes Faecalis

Alk

Alk

TSI agarNo GasNo H2S

UreaseNegative

Citrate agarPositive

SIM agarIndole Negative& No H2S

MRNegative

VPNegative

Nitrate ReductaseNegative (turned when zinc added)

Lysine DeaminationNegative

LysineDecarboxylation

Positive

Staphylococcus

saprophyticusCatalase: Positive

Yogurt, Food

& Water

microbiology

Yogurt

MicrobiologyHeat to boiling milk

Cool to 55ºC

Lactobacillus bulgaricus (Acidophile)

Streptococcus thermophiles (Thermophile)

Incubate @ 45ºC

Biochemistry: Fermentation (lactose) lactic acid (Lower pH)

Cow’s milk pH: 7

calcium caseinate (colloidal suspension)

calcium lactate free (soluble) casein

Food Microbiology

Salmonella & Shigella

Selective: Bile salts(select gram negative enteric bacteria)

Differentiate

Carbohydrate:

Lactose fermenters

pH: Neutral Red

H2S: ferric citrate

Escherichia coli

Salmonella shigella

Chicken Beef

Water MicrobiologyMPN “Most probable number”

Tube inside “Durham tube”

Carbohydrate: lactose broth

What is the pH indicator: Phenol Red

Small inverted tube present in the MPN tubes: Durham tubes

Presumptive Test

10ml 1ml 0.1ml

Sewage Water 3/3/3 = >1000 coliforms/ 100ml of water sample

Fecal coliforms

“Indicator organisms” water has

been contaminated with human or

animal feces

Obviously negative if PINK!!

Gas

Positive: Yellow

(Lactose fermentation)

+ Gas production

(Floating Durham)

Negative: Pink or

doesn’t float

ELISA testing &

Epidemiology

EPIDEMIOLOGY

InvestigationEpidemiologist Goals:

1.) Case of disease

2.) Route of Transmission

3.) Trace patter to identify original carrier “patient 0”

Test Tubes “Body Fluids”Phenol red infected??

Who

infected

me??

ELISA testing Testing Sample A & B

Before Antibodies are added:

(AG) Purified Antigen

Antibodies added:

Positive Control

Negative Control

Sample A

Sample B

After Antibodies are added:

(SA) Secondary Antibody Attach to the first antibody & has a chromogen attached

(SUB) Enzyme substrate detection reagent

(-) (-) (-) (+) (+) (+) (A) (A) (A) (B) (B) (B )