Outcome 2.4 Culture Techniques

Question 1

What are some limitations of sampling

Answer 1

If aseptic technique is not followed correctly then the sample may become contaminated by some feature of the sampling technique itself.

Normal flora can contaminate samples

Question 2

Name methods of sampling

Answer 2

Swabs, blood samples, urine samples, stool samples, cerebrospinal fluid, lavage

Question 3

Since bacteria have different _____, it is possible to use different ____ for screening and as an aid to identification

Answer 3

growth requirements

selective media

Question 4

Some viruses will grow ____ in culture. To identify clinical isolates, the specimen is used to____ in culture.

Virus can be isolated from the culture for
identification by although superseded by which are more definitive

Answer 4

• on human cells
• infect cells
• electron microscopy (EM),
• ELISA/ Immunodetection and PCR testing

Question 5

Nutrient agar media can be used for

Answer 5

General cultivation of bacteria

Question 6

Minimal media are those that

Answer 6

contain the minimum nutrients possible for colony
growth, generally without the presence of amino acids, and are often used by
microbiologists and geneticists to grow “wild type” microorganisms

Question 7

The basic principal behind selective media is that

Answer 7

they will support the growth of a certain type of bacteria while inhibiting the growth of other bacteria

Question 8

Describe Eosin Methylene Blue Agar

Answer 8

EMB is a selective media that contains dyes that are toxic for Gram positive bacteria and bile salt which is toxic for Gram negative bacteria other than coliforms. Only Gram negative coliform bacteria e.g. E. coli will grow on this media

Question 9

Describe Mannitol Salt Agar

Answer 9

Mannitol Salt Agar is a selective media that MSA contains a high concentration (about 7.5%-10%) of salt (NaCl), making it selective for Gram-positive bacteria (Staphylococcus and Micrococcaceae)
since this level of salt is inhibitory to most other bacteria

Question 10

Describe Mackonkey Agar

Answer 10

A differential media that contains bile salts (to inhibit most Gram-positive bacteria), crystal
violet dye (which also inhibits certain Gram-positive bacteria) and neutral red dye which turns pink if the microbes are fermenting lactose. The media is used
to selectively isolate Gram-negative and enteric (normally found in the intestinal
tract) bacilli and differentiate them based on lactose fermentation.

Question 11

5

Answer 11

5

Question 12

Describe Modified Thayer Martin Agar

Answer 12

A differential media which is supplemented to contain
5% chocolate sheep blood and antibiotics. It is used to isolate Neisseria species of bacteria which are responsible for causing meningitis
Antibiotics are used to which Neisseria bacteria are
usually resistant

Question 13

Describe Blood Agar

Answer 13

Is a differential media that distinguishes microbes based on their ability to lyse red blood cells (RBC) by the production of toxins

Question 14

Define differential media

Answer 14

allows the growth of different types of bacteria but also have some way of allowing the different bacteria that grow to be distinguished

Question 15

What is the principle of biochemical techniques

Answer 15

Bacteria produce an array of enzymes and the presence of these enzymes can be tested for

Question 16

Describe the coagulase test

Answer 16

Coagulase is an enzyme that clots blood plasma by
converting soluble fibrinogen to insoluble fibrin.
Membrane bound coagulase is detected using a slide test.
A drop of culture is added to a microscope slide followed bya drop of plasma. Clumping can be observed under the microscope
Used to distinguish coagulase (+) S. aureus and
coagulase (-) S. epidermidis or S. saprophyticus.

Question 17

Describe the Nitrate Reduction Test

Answer 17

The nitrate reduction test is usually carried out to aid in the identification of enteric bacteria which usually possess the ability to reduce nitrate to nitrite and other N-containing compounds.
he test is carried out by inoculating a Nitrate
Broth tube with the test organism and allowing the culture to grow. If the organism is Nitrate Reduction (+) then Nitrite should be detectable in the tube.
To detect the nitrite equal quantities of α-naphthylamine and sulphanilic acid
reagents are added to the tube. If nitrite is present then the solution will immediately turn red and the organism is positive for nitrate reduction.

Question 18

Describe the Indole test

Answer 18

Tests for the ability of a microorganism toproduce the enzyme tryptophanase. Thisenzyme breaks down tryptophan (amino acid)into indole, pyruvate and ammonium.
Bacteria are cultured in peptone containing broth and then tested with a few drops of Kovac’s Indole Reagent.
The presence of indole is determined by the development of a red film around the top of the
culture tube

Question 19

Describe the oxidase test

Answer 19

The oxidase test is used to distinguish the different members of the enteric bacteria (all (-)) from Pseudomonas sp. (all (+)). The test relies on the presence of oxidase enzymes which can oxidise a colourless compound to c red/pink coloured one.
The test can be carried out directly on colonies growing on an agar plate by adding a few drops of the reagent. If the reagent turns blue/purple then the test organism is Oxidase (+).