Direct Methods of Analysis.

Question 1

What are the 3 steps in the direct method of identifying bacteria?

Answer 1

Identify bacteria via microscopy.

Culture bacteria.

Carry out antimicrobial sensitivity test on cultures.

Question 2

What 8 steps are carried out when directly analysing bacteria?

Answer 2

Direct smear.

Culture the sample.

Analyse colony morphology.

Apply differential tests e.g.

Gram stain.

Perform preliminary tests e.g. oxidase or catalase tests.

Biochemical tests (conventional or commercial).

Antibiotic sensitivity.

Question 3

How do we perform a direct smear so we can view a sample under the microscope?

Answer 3

By smearing some of the sample onto a microscopic slide.

Question 4

Why do you stain samples before you view them under the microscope?

Answer 4

So it is easier to view under the microscope.

Question 5

How do you culture a bacterial sample?

Answer 5

You smear some of the sample onto a nutrient agar such as blood agar or MacConkey agar.

Question 6

How can growing a bacterial strain on a growth media help you to identify the strain of bacteria?

Answer 6

You can subject the agar and sample to different conditions.

E.g. by depriving it of oxygen etc.

Question 7

What bacterial feature is usually analysed during microscopy?

Answer 7

The morphology of the culture.

Question 8

What is a good example of a differential test that we can perform on bacteria?

Answer 8

The Gram stain which will tell us whether the bacteria are gram positive or gram negative.

Question 9

What are the 2 categories of biochemical tests that we can perform on a bacterial sample?

Answer 9

Conventional tests.

Commercial tests.

Question 10

What does antibiotic sensitivity testing allow us to do?

Answer 10

To see what medication works best against this bacteria.

Question 11

What are the 4 types of microscopy test that we can perform to help us identify an unknown bacteria?

Answer 11

Wet mount.

Gram stain.

Acid fast stain.

Fluorescence testing.

Question 12

What does the KOH test tell us about a bacteria?

Answer 12

If the bacteria uses oxygen.

Question 13

What is a positive KOH test?

Answer 13

Bacteria that utilise O2 will produce bubbles.

Question 14

What does the wet mount technique involve?

Answer 14

Placing the sample on a microscope slide with solution.

You then view the sample through the microscope.

Question 15

How is the Gram stain carried out?

Answer 15

By adding certain chemicals to the sample in a particular order.

Once the sample is placed under the microscope it will either be stained pink or blue.

Question 16

What colour do gram negative bacteria turn after being gram stained?

Answer 16

Pink is always gram negative.

Question 17

What colour do gram positive bacteria turn after being gram stained?

Answer 17

Blue is always gram positive.

Question 18

Why can there sometimes be both gram negative and gram positive bacteria in the same sample?

Answer 18

As there may be more than 1 type of bacteria in each sample.

Question 19

Acid fast stains are used to identify what kind of bacteria?

Answer 19

Mycobacterium tuberculosis.

Question 20

What does the fluorescence technique involve?

Answer 20

A specialised microscope which emits fluorescent light.

Question 21

How is the fluorescence technique carried out?

Answer 21

The sample is stained with a fluorescent dye that adheres to certain bacteria.

This allows us to see whether any of these bacteria are within the sample.

Question 22

What are the 8 steps of the Gram stain method?

Answer 22

Smear onto slide.

Air dry.

Heat fix.

Stain with crystal violet and leave for 1 minute.

Add iodine.

Rinse with ethanol. (10 seconds).

Rinse with water.

Stain with safranin.

Question 23

Culturing bacteria should only be done where?

Answer 23

In the lab.

Question 24

Why should bacteria be transferred to different media within a safety hood?

Answer 24

So they do not contaminate the immediate area or infect the person handling them.

Question 25

What are the 2 main types of media that we generally use to culture bacteria?

Answer 25

MacConkey’s agar.

Blood agar.

Question 26

What kind of growth media is MacConkeys agar?

Answer 26

A differential/selective media.

Question 27

What kind of growth media is blood agar?

Answer 27

An enriched non-selective media.

Question 28

What kind of bacteria will grow on blood agar?

Answer 28

Almost all bacteria.

Question 29

MacConkey’s agar will differentiate between what 2 types of bacteria?

Answer 29

Gram - and gram + bacteria.

Lactose fermenting bacteria or non-lactose fermenting bacteria.

Question 30

How does MacConkeys agar tell us if we have lactose fermenting bacteria?

Answer 30

It will turn pink.

Question 31

How does MacConkeys agar tell us if we have non-lactose fermenting bacteria?

Answer 31

It remains colourless.

Question 32

How does MacConkeys agar differentiate between Gram - and gram + bacteria?

Answer 32

Gram + bacteria will not grow on MacConkeys agar.

Question 33

Incubation of bacteria usually takes how long?

Answer 33

At least 48 hours.

Question 34

What are the 2 pigments that can be produced in a growth media?

Answer 34

An endopigment.

An exopigment.

Question 35

When is an endopigment produced in a growth media?

Answer 35

When a bacterial colony is placed into a differential media and only the bacterial colony changes colour.

Question 36

When is an exopigment produced in a growth media?

Answer 36

When a bacterial colony is placed into a differential media and the media itself changes colour.

Question 37

When does haemolysis of blood agar occur?

Answer 37

When the bacteria lyse the red blood cells in the blood agar.

Question 38

What are the 3 categories of haemolysis?

Answer 38

Organisms that cause partial haemolysis (alpha haemolysis).

Organisms that cause complete haemolysis (beta haemolysis).

Organisms that do not cause haemolysis (gamma haemolysis).

Question 39

How does the bacterial colony appear when alpha haemolysis has occurred?

Answer 39

The colony will appear to be dark green.

Question 40

How does the bacterial colony appear when beta haemolysis has occurred?

Answer 40

The area where the colony is located will appear light yellow.

Question 41

How does the bacterial colony appear when gamma haemolysis has occurred?

Answer 41

The area around the colony will be unchanged.

Question 42

How do we isolate salmonella from a fecal sample?

Answer 42

Sample is inoculated in Rappaport or selenite broth.

This only allows for salmonella to grow.