Biochemical Tests

Biochemical Testing
(Question 1) Staining provides information on bacterial morphology Staining provides little information as to genus and species of particular bacterium To identify bacteria, need to rely on biochemical testing Series of tests that provides a metabolic fingerprint of each organism

Biochemical Testing
(Question 2) Each different species has different genome (DNA)

Different species of bacteria will synthesize different series of protein enzymes Enzymes catalyze all of the various chemical reactions of which the organism is capable

Biochemical Testing
(Question 2) Different species of bacteria carry out different and unique sets of biochemical reactions In some cases, the difference is not in the gene content, but in regulation of expression of the gene

Biochemical Testing
(Question 3)

Key feature of biochemical tests is their ability to discriminate between bacteria More interested in general end result than in what specific enzymes gave that result
Ex: For carbohydrate metabolism, just want to know if it produced acid -- dont need to know specific end product or which enzyme(s) produced it

Biochemical Testing
(Question 3)

Sensitivity and timing important

Sensitivity should be sufficient to allow discrimination; Test result may vary with length of time that culture or test is incubated

Medium composition and incubation T also important Usually looking for color change

Biochemical Tests
What you need to know:
Cellular biochemical (enzymatic) reaction Identity of substrate the bacterium is using Identity of cellular product (what were testing for) Reagents used to test for product Positive and negative test results (what you see) Be able to answer questions associated with each test

Acid Production in Sugar Metabolism

Influenced by: (Question 4)
The sugar present
Does the bacterium ferment the sugar?

Peptone (protein peptone amino acids)

Does the bacterium utilize amino acids as its carbon source?

The pH range of the indicator

Does the bacterium produce acid/ammonia in the range of the indicator?

The pH of the medium

Is the medium buffered in a range suitable for the indicator being used?

Sugars Tested
Glucose Lactose Raffinose Sucrose

(Questions 5-7)
Indicator: Brom thymol blue Range: 6.0 (yellow) to 7.6 (blue)

Results:
A/G, acid (yellow) and gas (Durham tube) produced A, acid (yellow) produced (A), some acid produced -- lime green 0+, no acid (green) or gas produced; growth (peptone) 0+/B, no acid or gas; growth (peptone); alkaline (blue) 0-, no acid (green) or gas produced; no growth

Mannitol Fermentation
(Question 8) Medium: mannitol + beef extract + peptone Indicator: Brom cresol purple Range: 5.2 (yellow) to 6.8 (purple)

Results
A, acid produced (grayish yellow) 0+, no acid produced (purple); growth (peptone)

0-, no acid produced (purple); no growth

MacConkey Agar
(Questions 9 - 11)
Test for lactose utilization Indicator : Neutral red pH range: 6.8 (red) to 8.0 (yellow) Acidic pH gives more vivid color (on plate) than BTB Remember: Medium contains peptone
Difference in colony color makes this a differential medium.

MacConkey Agar
(Question 11)
Escherichia coli Salmonella typhimurium

Selective: Crystal violet and bile salts inhibit most Gram positives

Citric Acid Utilization

(Questions 12-14)
Citric acid: intermediate in TCA cycle Na-Citrate Acetic or succinic acid
Test: Can bacterium utilize citrate as its sole carbon source? (Can bacterium transport citrate across membrane?)

At pH 7, citric acid is present as Na+Citrate-. Oxidation of Citrate- to CO2 by TCA cycle releases Na+, which displaces NH4+ from ammonium phosphate. NH4OH forms to raise pH.

Citric Acid Utilization

(Question 15)
Indicator: Brom thymol blue
Results:

citrate +, any blue color citrate -, no color change

Methyl Red/Voges-Proskauer Medium

(Question 16)
Glucose + Peptone + Phosphate NO INDICATOR IN MEDIUM!
(added later)

Methyl Red Test

Test for fermentation of glucose to acid products (large quantities of acid)
Add indicator to 1 ml of culture

Incubation time important: Some bacteria degrade acid over time

MR +: red color MR -: yellow color

(pH < 5.2) (pH > 5.2)

Voges-Proskauer Test
(Questions 17 - 20)
Butanediol Fermentation Pathway: 4 pyruvates 2 acetoin + 4 CO2 2 acetoin + 2 NADH Enzymatic oxidation

+ 2 H+ 2 butanediol + 2 NAD+ Enzymatic reduction Chemical oxidation

VP reagents: butanediol + -naphthol + KOH + O2 acetoin VP I VP II

VP + = pink-red color VP - = brownish color Creatine intensifies color change. Possible results: MR-/VP-; MR+/VP-; MR-/VP+ (MR+/VP+, rare)

Enzymatic Esculin Hydrolysis

(Questions 21, 22)
HO O O

excreted
HO O O

carbon source
CH2 OH O HO OH OH OH

O CH2OH O HO OH OH

+
HO

Esculin
O

Esculetin
H O

b -D-Glucose
O O

HO

+
HO

Ferric salts

Fe3+

Fe3+ O H

Phenolic iron complex

Enzymatic Esculin Hydrolysis

(Question 22)
Esculetin + Fe3+-salts Phenolic iron complex (brown/black)

Bile salts: inhibits some Gram positives (selective)

Lysine Decarboxylase
(Questions 23, 24)
Indicator: Brom cresol purple, pH 5.2 - 6.8

Glucose Acidic products (yellow)

Enzyme synthesis

Acid

lysine decarboxylase (LD)

HHHHHO H2N-C-C-C-C-C-C-OH H H H H NH2 lysine

LD

HHHHH H2N-C-C-C-C-C-NH2 + CO2 HHHHH cadaverine

Lysine Decarboxylase
(Question 24)
Indicator: Brom cresol purple, pH 5.2 - 6.8

Glucose Acidic products (yellow)

Enzyme synthesis

Acid

lysine decarboxylase (LD)

HHHHHO H2N-C-C-C-C-C-C-OH H H H H NH2

LD

Carboxyl group

HHHHH H2N-C-C-C-C-C-NH2 + CO2 HHHHH

decarboxylates

Lysine Decarboxylase
(Question 24)
Indicator: Brom cresol purple, pH 5.2 - 6.8

Glucose Acidic products (yellow)

Enzyme synthesis

Acid

lysine decarboxylase (LD)

*May need to check pH.

HHHHHO H2N-C-C-C-C-C-C-OH H H H H NH2

LD

HHHHH H2N-C-C-C-C-C-NH2 + CO2 HHHHH alkaline (purple)

Lysine Decarboxylase
Cadaverine
Associated with the decay of organic matter Often found in gingival areas Associated with periodontal disease

Media is 1:5 mix of glucose:lysine (by mass)

Lysine in excess so amino acid utilization predominates Glucose needed to produce acid products that activate lysine decarboxylase

If media was 5:1 mix of glucose:lysine

- Glucose utilization predominates and so do acid products Get false negative

Indole Test
(Questions 25, 26)
Tryptone media: source of tryptophan tryptophanase Tryptophan Indole + Pyruvate
excreted Lactate (fermentation) or Acetyl CoA (respiration) Amino acids

+ NH3

Kovacs reagent: isoamyl alcohol

(extracts indole) p-dimethylaminobenzaldehyde (color reaction)

Kliglers Test
(Questions 27, 28)
Cysteine/methionine degradation H2S (No O2) Underground deposits of decaying plant material Present in wells drilled in shale or sandstone Coal or peat deposits (crude oil) Paper mills, petroleum refineries

Poisonous gas that can paralyze the respiratory system in high concentrations.

Phenol red: present but not used in ID

hydrogen sulfide

H2S + FeSO4 FeS + H2SO4

ferrous sulfate ferrous sulfide sulfuric acid

Primary indicator

black precipitate

Urease Test
(Question 29)
H2N H2N C O + 2 H2O CO2 + H2O + 2 NH3 (NH4)2CO3
Amino acids ammonium carbonate (alkaline)

urea

Indicator: phenol red Buffer: phosphate (to prevent weak positive) Left: Urease + (pinkish-red) Right: Urease - (yellow-orange)

Electron Transport System (ETS)

Fp = flavoprotein Fe-S = iron-sulfur Q = quinone Cyt = cytochrome
2 H+
2 e-

2 H+

OUT

2 e-

Fp

Fe-S Q
2 e-

IN
NADH + H+ FADH2

Cyt b
2 e-

2 H+ 3 H+ + 3 OH3 H2O

H+

Cyt o

3 H+ + 1/2 O2

H2O

Catalase Activity
(Questions 30, 31)
2H2O2

catalase

2H2O + O2

By-product of respiration Could damage DNA

Add 3% H2O2 to smear of cells

Bubbles (O2)

Aerobes have ETS and need catalase Some anaerobes have it (Bacteroides)

Anaerobic Respiration
Fp = flavoprotein Fe-S = iron-sulfur Q = quinone
2 H+

OUT

2 e-

Fp

2 H+
2 e-

Fe-S Q
2 e-

IN
NADH + H+ FADH2

Cyt b
2 e-

2 H+ 3 H+ + 3 OH3 H2O

Different enzyme

Nitrate reductase

NO3- + 2 H+ (N = +5) nitrate terminal electron acceptor

NO2- + H2O (N = +3) nitrite

Nitrate Reductase
(Questions 32 - 34)
nitrate

NO3- + 2 H+ + 2 e- H2O + B

nitrite

NO2-

NO, N2O, A NH2OH, NH3, N2

Step 1: Test for nitrite

NO2- + Rgt I & II HNO2 + iodide iodine + starch blue (+1)

Step 2: Test for nitrate NO3- + Zn (s) NO2blue (-)
Cells must be grown anaerobically (O2 interferes) Negative result after Step 1:

A B

Nitrite further reduced by bacteria No color after Step 2 (+2) Nitrate not reduced Blue color after Step 2 (-)

Bacterial Growth

Consider the impressive growth potential of bacteria -- what they do best is grow fast. No other form of life can grow as fast as bacteria. Thus given nutrients, bacteria are the first to exploit any environment.

Environmental Factors Affecting Growth Rate

(Question 35)

Culture medium Temperature pH Solutes (osmotic pressure) Oxygen Water availability

Bacterial Growth Curve

(Question 36)

Determination of Doubling Time

(Questions 37, 38)

When y = 2 y0 t - t0 = g g=4-2=2h

Log10

y
y0 t0 t

g = 1/k
k = growth rate constant