Identifying Bacteria

Question 1

Cell morphology (shape)

Answer 1

Staining
Shapes of bacteria - important for identification and microscopic observation
The cell wall maintains the shaoe

Question 2

Stains are used for what

Answer 2

• Make it Easier to see bacteria
• Show structures such as spores and flagella
• Differentiate different biochemical properties of bacteria

Question 3

What are the 4 modes on a light microscopes

Answer 3

Bright field
Phase contast
Dark field
Fluorescence

Question 4

Gram negative colour

Answer 4

Red or pink

Question 5

Colony morphology

Answer 5

Bacteria form colonies with consistent characteristics e.g. shapes, textures and colours
Colony morphology is helpful when spotting contamination and can be used in preliminary identification.
You can compare organisms when they’re grown on the same media and under the same conditions

Question 6

What are the different form, elevation and margin and the show how theh look

Answer 6

Form- circular, irregular, filamentous, rhizoid
Elevation- raised,convex, flat, umbonate, crateriform
Margin- entire, undulate, filiform, curled, lobate

Question 7

Cell morphology (shape)

Answer 7

Generally requires staining
Shapes of bacteria are important for identification and microscopic observation.
Cell wall maintains shape of bacteria
Chains and groups occur when bacteria remain attached to each other after cell division

Question 8

Cellular structures

Answer 8

Used to aid identification of flagella, spores, capsules.
Staining used to visualise
E.g. salmonella typhi

Question 9

Staining

Answer 9

Bacteria are very slow and very difficult to see even using a microscope.
Stamina are used to make bacteria easier to see,
Show up structures such as spores and flagella,
And to differentiate biochemical properties of bacteria.

Question 10

Gram stain

Answer 10

Gram stain divides most clinically significant bacteria into two main groups which are

• Gram +ve and -ve
• it is the first step in bacterial identification

Question 11

Visualisation for gram staining compound

Answer 11

Use light microscopes 
LM has 4 modes - 
Bright field 
Phase contrast 
Dark field 
Fluorescence 
-Bright field used for observing differences in density 
-Standing improves contrast

Question 12

Colour of gram positive

Answer 12

Purple
Gram +ve rod
Gram +ve cocci

Question 13

Colour of gram negative

Answer 13

Red or pink

Gram- ve rod

Question 14

Acid fast stain

Answer 14

Used for bacteria that do not gram stain
Used for mycobacteria sp e.g. M. Tuberculosis
Once stained cells cannot be decolourised by weak acid/ethanol
The waxy capsule of acid-fast organism take up and retain red dye carbolfuchsin

Question 15

Phase contrast and dark field

Answer 15

Gram staining kills cells
Phase contrast and dark field allow visualisation of live cells
Phase contrast observes differences in refractive index
Dark field applies light from the sides and scattering is observed.

Question 16

Fluorescence microscopy

Answer 16

Flureosense can be used to visualise specimen, some specimens will fluoresce naturally e.g. chlorophyll
Other require a stain which will fluoresce e.g.
Diamidino 2 phenylindole (DAPI)
molecular probes for specific genetic staining

Question 17

Electron microscopy

Answer 17

Fires an electrical beam at a sample stained with gold.
Electrons bounce of the specimen and are processed
-SEM (scan across the surface)
Has Very high resolution and maginification
- electromagnets can transmit electrons into sample (TEM)

Question 18

How to visualise specimen in 3D

Answer 18

Atomic force microscopy
- a small stylus is positioned close to the sample and the repulsive forces from the sample as the stylus moves up and down the hills and valley and then the computer processes the responses into a 3D image

Question 19

Confocal laser microscopy

Answer 19

Laser coupled to light microscope
Scans individual layers of the sample
Can also stain the various components of a sample to distinguish between them

Question 20

Biochemical tests

Answer 20

Use broth or solid media impregnated with specific nutrients and chemical indicators for specific products mainly changes in pH.
Looking for production of acid and or gas

Question 21

Sugar fermentation

Answer 21

Sugar= acid + gas
=is suppose to be an arrow which is fermentation
Acid lowers pH
And gas is not always produced
The media contains pH indicator to detect acid production and inverted durhams tube to collect gas
Before it is pink and if it consumes it it produces acid and changes pH which changes colour to red

Question 22

Other ways to characterize an organism

Answer 22

Is it aerobic or anaerobic growth maybe both?
Food sources?
Nitrogen sources?
Presence of certain enzymes ?

Question 23

Does it require oxygen to do acid production test how to check

Answer 23

Growth observed in stab culture
Heat stab wire with bunsen burner and stab it through.
Growth is observed by colour change of bromothymol blue.

Question 24

Catalase production

Answer 24

Enzyme that catalyse decomposition of hydrogen peroxide to oxygen and water
Formed by most aerobic bacteria
Observe gas formation when bacteria is exposed to hydrogen peroxide
This will test if it has catalyse or not

Question 25

Oxidase test

Answer 25

Required by bacteria that have respiratory chain Indicates the presence of cytochrome oxidase If it has cytochrome oxidase indicator turns purple if not then it just the natural colour

Question 26

Coagulase tests

Answer 26

Later aggultinin tests for coagulase postive staphylococcus sp Latex breads coated with fibrinogen Forms a clot when plasma protein is added to coagulase-positive

Question 27

Metabolic indicator media

Answer 27

Used to indicate specific metabolic abilities of isolates Type of biochemical test Reflect the presence of specific enzymes e.g. urease, citrate utilisation, haemolysis

Question 28

Example of a metabolic indicator media

Answer 28

Some bacteria e.gm proteus vulgaris process the enzyme urease Can convert urea to ammonia and co2 Urease production detected on christensens urea agar Christensens agar contains a pH indicator because we are producing ammonia and ammonia is alkali.whixh is phenol red Yellow at pH 6.8 and red at pH 8.4 Ammonia raises the pH Urease positive =red Negative if doesnt contain urea enzyme would be light redish pink but if its positive it would be bright pink

Question 29

Haemolysis

Answer 29

It is the Breakdown of blood It uses blood agar to distinguish between pathogenic bacteria - streptococcus pneumoniae = a - haemolytic - streptococcus pyrogenes = B - haemolytic

Question 30

What is alpha haemolysis

Answer 30

A zone of partial destruction of tbc around colonies with a greenish to brownish discoloration of the medium

Question 31

What is beta haemolysis

Answer 31

Clear colourless zone of complete rbc discoloured

Question 32

What is no haemolysis

Answer 32

No haemolysis activity or discoloration of rbc around colonies

Question 33

Why do all they mediator tests

Answer 33

To make a flow chart to understand

Question 34

Why use serotyping

Answer 34

Allows classification of microorganisms to the sub species level based on the antigens they possess. Use this for Salmonella has over 4400 serotypes e.g. salmonella enterica. Use this for Vibrio cholerae has 139 serotypes where only two of them are pathogenic

Question 35

Example of serotyping

Answer 35

Salmonella- agglutination testing to test for different salmonella serotypes It is based upon variants of somatic (o) and flagellar (H) antigens. Useful because we have a agglutination test for the o antigen. But salmonella can have two different H types. So we use seven gard medium to allow the organism to express that second H antigen in phase 2

Question 36

What is O antigen

Answer 36

It is the side chains of repeating sugar units which project through the outer lipo- polysacharide layer of the cell wall. There are over 60 different o antigens It is heat stable (100 degrees for 2.5 hours) It is alcohol stable

Question 37

What is H antigen

Answer 37

It is the flagellar protein It is both heat and alcohol labile The flagella will come apart at 100 degrees for 30 mins. -has di phasic production so they can flip between 2 so get to express one then the other allows us to have different sets of antigens

Question 38

Test for salmonella serotyping

Answer 38

Take your sample ans put into a test well which has agglutination antisera in it. So each one of the well will have the opposite to the antigen. The antigen is the key. Each test well has a different lock and each lock is different and you get see when antigen fits in the lock. -Add 1 drop of anisteria onto a slide for agglutination and add a pure colony then mix reagents thoroughly and Rock the slide in the circular motion for 30 secs and observe aggly6

Question 39

3 main types of DNA

Answer 39

16sRNA gene Ribotyping Pulsed field gell electrophoresis

Question 40

16s rRNA

Answer 40

Every bacteria has this 16s rRNA gene | It need to be able to produce ribosomes

Question 41

Ribtyping

Answer 41

Used to identify clostridium difficile. This is an anaerobic organism. You look at the space region between 16s and 23s rRNA regions. C. Difficile possess multiple copies of the rRNA genes. It can vary in size between copies of the same genome 250 to 600 base pairs. It can vary number between strains

Question 42

Pulsed field gel electrophoresis

Answer 42

``` It is equivalent to DNA finger printing used to track disease outbreaks Used for e.g. Campylobacter jejuni Salmonella sp Shigella sp Yersinia posits Listeria monocytogenes Escherichia coli ```

Question 43

How to do pulsed field gel electrophoresis

Answer 43

Genomic DNA is extracted. Restriction enzymes cut genomic DNA into a small number of fragments usually 10-20. Resolved by PFGE whicj is constantly changing the direction of the electrical field during electrophoresis. Differential migration of large DNA fragments through agarose gels. Then look at Fragments profile to see what specific specifies/ subspecies it is

Question 44

Pulsed field gel electrophoresis process

Answer 44

Take bacterial cells from an agar plate and mix bacterial cells with melted agarose and pour into a plug mold. The bacterial cells are broken open with biochemical or lysed so that the DNA is free in the agarose plugs. Load the DNA gelatin plug into a gel and place it in an electric field that separates DNA fragments according to its size. The gel is stained so that DNA can be seen under UV light. A digital camera takes a photograph of the gel and stores the picture in the computer. Then you see what the profile looks like and see the variations.

Question 45

Phase typing

Answer 45

Take unknown organism and streak it out onto a plate and then split it into however many u want and then on the surface of the plate apply 4 different bacterial viruses. The virus if it can will effect the organism and form plaques which is a gap where there is no grow because the virus has killed the bacteria. Compare the cross organisms to see what phages it is susceptible too.