Question

1. In the unique bacterial enzyme laboratory (e.g., DNAse, Starch, NO3 tests) there were a number of tests that required the addition of some reagent after the cells had grown. List and describe all tests with reagents added after growth. Why do we have to add these reagents after incubation, when in many other tests the indicators are incorporated into the medium? Be sure that you know how to score a reaction positive or negative, and what each of those results mean. Do you have to test for intracellular and extracellular enzymes differently? Be sure to know at least one example of the test used for each of these different enzyme reactions.

Answer 1

Tests used to identify Gram Positive Bacteria

• Catalase Test
• Mannitol Salt Agar (MSA)
• Blood Agar Plates (BAP)
  • Streak-stab technique
• Taxos P (optochin sensitivity testing)
• Taxos A (bacitracin sensitivity testing)
• CAMP Test
• Bile Esculin Agar
• Nitrate Broth
• Spirit Blue agar
• Starch hydrolysis test
• Motility Agar
• Coagulase Test

Tests used to identify Gram Negative Bacteria

• Oxidase Test
• Sugar (eg glucose) broth with Durham tubes
• Methyl Red / Voges-Proskauer (MR/VP)
• Kliger’s Iron Agar (KIA)
• Nitrate Broth
• Motility Agar
• MacConkey agar
• Simmon’s Citrate Agar
• Urease test
• Sulfur Indole Motility Media (SIM)

DNAase test

DNA hydrolysis test or Deoxyribonuclease (DNase) test is used to determine the ability of an organism to hydrolyze DNA and utilize it as a source of carbon and energy for growth.An agar medium; DNase agar, a differential medium is used to test the ability of an organism to produce deoxyribonuclease or DNase.

This medium is pale green in color because of DNA-methyl green (indicator) complex (Note: Methyl green is a cation which binds to the negatively-charged DNA). It also contains nutrients for the bacteria.

If the organism that grows in the medium produces Deoxyribonuclease, it breaks down DNA into smaller fragments. When the DNA is broken down, it no longer binds to the methyl green, and green color fades and the colony is surrounded by a colorless zone...

Requirements:

1. Media: DNase Agar or DNase agar with Methyl green indicator.
2. Reagent: Hydrochloric acid (1mol/L) only when DNase agar without indicator is used..

1. Positive: When DNA is hydrolysed, methyl green is released turning the medium colorless around the test organism.
2. Negative: If there is no degradation of DNA, the medium remains green.

Test results

1. DNase Test positive organisms:
   1. Serratia marcescens
   2. Staphylococcus aureus
   3. Campylobacter jejuni (some strains)
   4. M. Catarrhalis
2. DNase test negative organisms:
   1. Staphylococcus epidermidis
   2. Neisseria gonorrhoeae

Uses of DNase Test:

1. It is used to differentiate S.aureus (DNase +ve) from other Staphylococci that do not produce such enzyme. The DNase test is particularly useful when plasma is not available to perform a coagulase test or when the results of a coagulase test are difficult to interpret.
2. DNase test distinguishes M. catarrhalis from all other gram-negative diplococci (e.g. Neisseria gonorrhoeae & Neisseria meningitidis) of human origin.

Limitation of DNase Test

1. Some MRSA strains do not give positive DNase test result and some strains of the coagulase-negative staphylococci such as Staphylococcus capitis may give weak reactions.
2. Serratia and Moraxella species also produce deoxyribonuclease.
3. 1N HCl is bactericidal for Staphylococci. Once the HCl has been applied, the test must be read within 5 minutes and cannot be continued by re-incubation....

Starch test:-  Many bacteria produce extracellular enzymes used to catalyze chemical reactions outside of the cell. In this manner, nutrient sources, such as starch, that are too large to be absorbed through the cell membrane can be broken down into smaller molecules and transported into the cell via diffusion...

In the starch hydrolysis test, the test bacteria are grown on agar plates containing starch. If the bacteria have the ability to hydrolyze starch, it does so in the medium, particularly in the areas surrounding their growth while the rest of the area of the plate still contain non-hydrolysed starch. Since no color change occurs in the medium when organisms hydrolyze starch, iodine solution is added as an indicator to the plate after incubation. While the non-hydrolysed starch forms dark blue color with iodine, its hydrolyzed end products do not acquire such dark blue color with iodine.

Consequently, transparent clear zones are formed around the colonies that hydrolyze starch while the rest of the plate show a dark blue coloration as iodine forms the colored complex with starch.

Media: Starch agar is a simple nutritive medium with starch added. Beef extract and pancreatic digest of gelatin provide nitrogen, vitamins, carbon and amino acids. Agar is the solidifying agent and starch is the carbohydrate.

Composition:- Peptic digest of animal tissue 5.000, Sodium chloride 5.000, Yeast extract 1.500, Beef extract 1.500 Starch, soluble 2.000 Agar 15.000 Final pH ( at 25°C) 7.4±0.2...

Expected Results

• Positive test:A clear zone around the line of growth after addition of iodine solution indicates that the organism has hydrolyzed starch.
• Negative test:A blue, purple, or black coloration of the medium (depending on the concentration of iodine..

Uses

• It aids in the differentiation of species of genera Corynebacterium, Clostridium, Bacillus, Bacteroides, Fusobacterium, and members ofEnterococcus spp.

Limitations

• It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
• Colonies cannot be subcultured from the medium after the addition of Gram’s iodine due to the oxidative nature of the reagent and the resulting cell death.....
• 
  STARCH HYDROLYSIS TEST Positive Negative No zone formation Zone of hydrolysis
  

NO3 test:-

Anaerobic metabolism requires an electron acceptor other than atmospheric oxygen (O2). Many gram-negative bacteria use nitrate as the final electron acceptor. Nitrate reduction test is a test that determines the production of an enzyme called nitrate reductase, which results in the reduction of nitrate (NO3)......Bacterial species may be differentiated on the basis of their ability to reduce nitrate to nitrite or nitrogenous gases....

Nitrate NO3 Nitrate reductase + Sulfanilic acid (Reagent A) Nitrite NO, Colorless (Nitrate-Sulfanilic acid) Nitrite reductase + a-naphthylamine (Reagent B) Nitric Oxide NO Nitric Oxide reductase Red precipitate (Prontosil) Nitrous Oxide N, Nitrous Oxide reductase Nitrogen N2

Media:

Nitrate Broth

Peptone 5 g/l, Meat extract 3 g/l, Potassium nitrate 1 g/l.

Expected Results

• Positive Test:
  – Development of a cherry red colouration on addition of reagent A and B
  – Absence of a red color development on adding Zn powder
• Negative Test:
  – A development of red color on addition of Zn powder..

• Uses

• All members of the Enterobacteriaceae family reduce nitrate, but some members further metabolize nitrite to other compounds. It is thus used to differentiate members of Enterobacteriaceae that produce enzyme nitrate reductase from Gram negative bacteria that do not produce the enzyme nitrate reductase.
• The reduction of nitrate may be coupled to anaerobic respiration in some species.
• It is used in differentiating Mycobacterium
• Identifying species of Neisseriaand separating them from Moraxella and Kingella The nitrate reduction test is a critical test for differentiating between N. gonorrhoeae and K. denitrificans, particularly when strains of K. denitrificans appear to be gram-negative diplococci in stained smears.
• Facilitating species identification of Corynebacterium

• Limitations

• The nitrate reduction test may be used as an aid in the identification of bacteria. Additional biochemical testing using pure culture is recommended for complete identification.
• Due to the possible presence of nitrite in the culture media, a low nitrite media such as Nitrate Agar or Nitrate Broth should be used for the nitrate reduction test.
• A negative zinc reduction (no color change) test, in combination with a negative nitrite reaction, is presumptive indication that the nitrate was reduced beyond the nitrite stage. Although a very common end product of nitrite reduction is nitrogen gas, other end products may be formed. Additional testing may be required to determine the final end products of the reaction.
• To avoid false-negative nitrite reduction reactions, negative nitrite reactions must be verified by the addition of zinc dust to the medium.
• Excess zinc dust has been reported to cause false-positive nitrite reduction reactions due to complete reduction of previously unreduced nitrate to ammonia.
  reduction no reduction to NO2 uninoculated ZINC DUST reduction to N2 NITRATE
  

Lipase test:- to identify bacteria capable of producing the exoenzyme lipase....Tributyrin agar is a differential medium that tests the ability of an organism to produce an exo-enzyme, called lipase that hydrolyzes tributyrin oil. Tributyrin agar is prepared as an emulsion so that the agar will appear opaque...

Tributyrin is too large to enter the cell, so a lipase is released to break it down prior to cellular uptake.

Media Used in Lipase TestT

Tributyrin agar: Peptic digest of animal tissue 5.0 gm/L, Yeast extract 3.0 gm/L, Agar 15.0 gm/L, Final pH ( at 25°C) 7.5±0.2...

Result Interpretation of Lipase Test

Positive Lipase test: clear zone around the bacterial growth

Negative Lipase test: absence of clear zone around the bacterial growth...

Staphylococcus aureus , positive, clear zone around colony

Clostridium perfringens negative, absence of clear zone around colony

Clostridium sporogenes positive, clear zone around colony....

Limitation:-

• Zones of opacity are small with non-glucose-fermenting rods.
• Some microorganisms may require up to 1 week to produce a positive lipase reaction.

Indole test

In this test, the organism under consideration is grown in peptone water broth. It contains tryptophan, which under the action of enzyme tryptophanase is converted to an Indole molecule, pyruvate and ammonium. The indole is then extracted from the broth by means of xylene. To test the broth for indole production, Kovac's reagent . Kovac's reagent consist of amyl alcohol and para-dimethylaminobenzaldehyde and concentrated hydrochloric acid. Kovac's reagent is actually used to determine ability of an organism to separate indole from amino acid tryptophan and it is added after incubation. A positive result is indicated by a pink/red layer forming on top of the liquid....

note:- yes we have tests for extracellular and intracellular enzymes differently... All the parts of this question has been answered above thats why i ket amswers a little elaborative... Plz read it carefully.. thank u