Identifying Bacteria Flashcards
Cell morphology (shape)
Staining
Shapes of bacteria - important for identification and microscopic observation
The cell wall maintains the shaoe
Stains are used for what
- Make it Easier to see bacteria
- Show structures such as spores and flagella
- Differentiate different biochemical properties of bacteria
What are the 4 modes on a light microscopes
Bright field
Phase contast
Dark field
Fluorescence
Gram negative colour
Red or pink
Colony morphology
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
What are the different form, elevation and margin and the show how theh look
Form- circular, irregular, filamentous, rhizoid
Elevation- raised,convex, flat, umbonate, crateriform
Margin- entire, undulate, filiform, curled, lobate
Cell morphology (shape)
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
Cellular structures
Used to aid identification of flagella, spores, capsules.
Staining used to visualise
E.g. salmonella typhi
Staining
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.
Gram stain
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
Visualisation for gram staining compound
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
Colour of gram positive
Purple
Gram +ve rod
Gram +ve cocci
Colour of gram negative
Red or pink
Gram- ve rod
Acid fast stain
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
Phase contrast and dark field
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.
Fluorescence microscopy
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
Electron microscopy
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)
How to visualise specimen in 3D
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
Confocal laser microscopy
Laser coupled to light microscope
Scans individual layers of the sample
Can also stain the various components of a sample to distinguish between them
Biochemical tests
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
Sugar fermentation
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
Other ways to characterize an organism
Is it aerobic or anaerobic growth maybe both?
Food sources?
Nitrogen sources?
Presence of certain enzymes ?
Does it require oxygen to do acid production test how to check
Growth observed in stab culture
Heat stab wire with bunsen burner and stab it through.
Growth is observed by colour change of bromothymol blue.
Catalase production
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
Oxidase test
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
Coagulase tests
Later aggultinin tests for coagulase postive staphylococcus sp
Latex breads coated with fibrinogen
Forms a clot when plasma protein is added to coagulase-positive
Metabolic indicator media
Used to indicate specific metabolic abilities of isolates
Type of biochemical test
Reflect the presence of specific enzymes e.g. urease, citrate utilisation, haemolysis
Example of a metabolic indicator media
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
Haemolysis
It is the Breakdown of blood
It uses blood agar to distinguish between pathogenic bacteria
- streptococcus pneumoniae = a - haemolytic
- streptococcus pyrogenes = B - haemolytic
What is alpha haemolysis
A zone of partial destruction of tbc around colonies with a greenish to brownish discoloration of the medium
What is beta haemolysis
Clear colourless zone of complete rbc discoloured
What is no haemolysis
No haemolysis activity or discoloration of rbc around colonies
Why do all they mediator tests
To make a flow chart to understand
Why use serotyping
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
Example of serotyping
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
What is O antigen
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
What is H antigen
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
Test for salmonella serotyping
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
3 main types of DNA
16sRNA gene
Ribotyping
Pulsed field gell electrophoresis
16s rRNA
Every bacteria has this 16s rRNA gene
It need to be able to produce ribosomes
Ribtyping
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
Pulsed field gel electrophoresis
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
How to do pulsed field gel electrophoresis
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
Pulsed field gel electrophoresis process
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.
Phase typing
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.
