Untitled Deck Flashcards
what colour do B-glucuronidase E.colicolonies grow?
Blue colonies
What media is used for B-glucuronidase E.coli?
Tryptone-bile-glucuronide-medium (TBX)
What is the procedure for the inoculation and incubation of B-glucuronidase E.coli?
1.) Using a sterile pipette transfer 1ml of test sample to a petri dish or 1ml of the initial dilution.
2.) Inoculate 2 plates per dilution.
3.) Repeat procedure with further decimal dilutions, using a new sterile pipette for each dilution.
4.) Pour into each petri dish 15ml of TBX medium, previously cooled to 44deg in the water bath.
5.) Carefully mix the inoculum with the media with horizontal movements and allow the media to solidify.
6.) The time lapsed between the addition of the inoculum and medium should not exceed 15 minutes.
7.) Invert the inoculated dishes so that the bottom is upmost and place in an incubator set at 44deg for 18-24h. The total incubation time should not exceed 24h.
What is the maximum number of colonies for B-glucuronidase E.coli allowed for a plate to be countable?
300
What is the minimum valid result for B-glucuronidase E.coli?
For a valid result it is necessary to count the CFU on at least 1 dish containing a minimum of 15 blue CFU.
How was the minced beef sample prepared?
1.) 25g of the food sample was placed into a stomacher bag.
2.) 225ml of MRD and 1ml of Tween was added.
What is MRD?
MRD is a diluent designed to maintain organisms by protecting the cells from unnecessary physiological shock that may occur using other aqueous solutions.
What is the purpose of TWEEN?
TWEEN solubilises any fat so that any bacteria can be released from it.
What is the confirmatory test for enterobacteriacea?
Oxidase and fermentation of sugars
What is the principle of the oxidase test?
Using a platinum wire, take a portion of each well isolated colony and streak onto a filter paper moistened with the oxidase reagent.
Or onto a commercially available stick or disk.
Consider the test to be negative when the colour of the filter paper does not turn dark purple within 10s.
What is the principle of the sugar fermentation test?
Using a wire stab the same colonies that gave a negative oxidase test into tubes containing glucose OF medium.
Overlay the surface of the medium with 1cm of sterile mineral oil.
If a yellow colour develops throughout the contents of the tube, reaction is regarded as positive.
What are the confirmatory tests for E.coli
Indole, Methyl red
What is the principle for the indole test?
Some bacteria split amino acid tryptophan into indole and pyruvic acid
* Indole can be detected with Kovac’s reagent
* Indole reacts with aldehyde in the reagent - pink/red colour which concentrates as a ring at the top
* Bacterium to be tested is inoculated in peptone broth (overnight at 37°C)
* Add few drops of Kovac’s reagent
* Pink ring = Indole positive
* No colour change = Indole negative
What is the purpose of the methyl red test
Two tests used to determine pattern of glucose metabolism of Enterobacteriaceae
* All Enterobacteriaceae metabolise glucose – initially produce pyruvic acid
* Some convert this through a mixed acid pathway – producing lactic, acetic or formic acid
* E. coli ferments glucose by this route
Red: Positive MR (E. coli)
No colour change: Negative
VP (E. coli)
What is the composition of TBX agar?
Enzymatic Digest of Casein,
Bile Salts,
X-Glucuronide,
Agar
What is the purpose of X-glucuronide in TBX agar?
The chromogenic agent X-glucuronide helps detect glucuronidase activity. E. coli cells absorb x-glucuronide and the intracellular glucuronidase splits the bond between the chromophore and the glucuronide. The released chromophore gives blue coloration to the colonies.
What agar does enterobacteriaceae grow on?
VRBG agar
What are 2 characteristics of enterobacteriaceae?
They ferment glucose and lactose and show a negative oxidase test.
What are the components of VRBG agar?
Peptone,
Yeast extract
NaCl
Bile salts (inhibit gram positive bacteria, except Enterococcus faecalis)
Glucose (carbon source)
Neutral red (pH-indicator)
Crystal violet (inhibit gram positive bakteria)
Agar
Water
What colour are the colonies on VRBG agar and why?
Colonies will appear as purple/pink with a halo. The bacteria will ferment glucose and carbon dioxide The halo is due to the trapped CO2 from the second layer of agar poured onto the petri dish. Bacteria will rapidly ferment the glucose and reduce the pH of the medium. When the medium becomes acidic the neutral red will turn the colonies will turn a pink/purple colour.
Explain any selective or differential ingredients in VRBG agar?
The bile salts are a selective ingredient which inhibit gram positive bacteria. The crystal violet will also inhibit gram positive bacteria. The glucose gets rapidly fermented by the bacteria which reduces the pH of the medium, this makes the media acidic. Neutral red in acidic environment will turn the colonies pink/purple.
What is the procedure for the inoculation and incubation of Enterobacteriacea?
1.) Using a sterile pipette transfer 1ml of test sample to a petri dish or 1ml of the initial dilution.
2.) Inoculate 2 plates per dilution.
3.) Repeat procedure with further decimal dilutions, using a new sterile pipette for each dilution.
4.) Pour into each petri dish 15ml of VRBG medium, previously cooled to 44deg in the water bath.
5.) Carefully mix the inoculum with the media with horizontal movements and allow the media to solidify.
6.) The time lapsed between the addition of the inoculum and medium should not exceed 15 minutes.
7.) After complete solidifcation of the mixture, add a covering layer of VRBG agar to prevent spreading growth and to achieve a semi-anaerobic condition. Allow to solidify.
8.) Invert the inoculated dishes so that the bottom is upmost and place in an incubator set at 37deg for 24h.
Explain the process for counting and selection of colonies of enterobacteriacea?
1.)Characteristic colonies are pink/purple with/without precipitation halos.
2.)Select the dishes which contain less than 300 colonies and count these colonies.
3.)Then at random choose 5 colonies to be subcultured on nutrient agar.
Explain the process for subculturing the selected colonies of enterbacteriacea ?
1.) Streak the colonies onto the non-selective. This will allow well isolated colonies to develop.
2.) Incubate at 37deg for 24h.
3.) Select a well isolated colony from each of the incubated plates for the biochemical confirmation tests.
Explain the biochemical confirmation tests for enterobacteriacea?
Oxidase test:
1.) Using a platinum wire , take a portion of each well isolated colony and streak onto a filter paper moistened with the oxidase reagent.
2.) Consider the test to be negative when the colour of the filter paper does not turn dark purple within 10s.
Fermentation of Sugars
3.) Using a wire stab the same colonies which gave a negative oxidase test into tubes containing glucose OF medium.
4.) Overlay the surface with 1cm of sterile mineral oil and incubate at 27deg for 24h.
5.) If a yellow colour develops throughout the content of the tube , regard the reaction as being positive.
What is the inoculation and incubation period for S.aureus?
1.) Transfer by means of pipette 0.1ml of the of the test sample to a baird parker agar plate.
2.) Repeat the procedure for further dilutions for enumeration techniques if necessary.
3.) If for certain products it is desirable to count low numbers of coagulase negative staphylococci, the level of detection can be raised by a factor of 10 by inoculating 1ml of the test sample onto a BPA plate.
4.) Carefully spread the inoculum as quickly as possible over the surface of the agar plate, trying not to touch the sides of the petri dish, using the spreader.
5.) Allow plates to dry with their lids on for about 15mins @RT.
6.) Invert the dishes prepared and place in an incubator at 34 deg for 24h. Then re-incubate for a total of 48h.
What should colonies presumed to be coagulae positive staphylococci look like?
Typical colonies should be black or grey, shining and convex 1-1.5mm after 24, 1.5mm-2.5mm after 48h and are surrounded by a clear partially opaque zone. After incubation for at least 24h, an opalescent ring immediately in contact with the colonies can appear in this zone.
Explain the process for the tube coagulase test?
1.) From the surface of each selected colony, remove an inoculum with a sterile wire and transfer it to a tube of BHI broth.
2.) With the same broth spread the suspension on a non-selective medium and incubate at 34deg for 24h to check the purity of the selected colony. Incubate broth in water bath at same conditions.
3.) Aseptically add 0.1ml of each culture to 0.3ml of the rabbit plasma in sterile haemolysis tubes and incubate at 34deg.
4.) By tilting the tube, examine for clotting f the plasma after 5h of incubation. If the test is negative re-incubate for 24h and re-examine.
5.) Consider the test to be positive if the cultures yield at least 3+ coagulase reactions.
6.) As a negative control for each batch of plasma, add 0.1ml of sterile BHI broth to the recommended amount of rabbit plasma and incubate. For the test to be valid the negative control should show no signs of clotting.
What does BPA contain?
Pancreatic digest of casein
Meat extract
Yeast extract
Sodium pyruvate 10.0
L-glycine
Lithium chloride
Potassium Tellurite
Egg Yolk Emulsion
Sulfamethazine solution
Agar
What are the selective ingredients for BPA?
Sodium pyruvate enhances the growth of S.aureus. L-glycine and potassium tellurite are selective ingredients which inhibit most other bacteria other than S.aureus. Potassium tellurite is a differential agent which is reduced to metallic tellurium by S.aureus resulting in black colonies. Egg yolk emulsion provides for demonstration of the proteolytic action of coagulase positive staphylococci shown by a clear zone.
Explain qPCR?
A laboratory technique used to amplify and simultaneously quantify a targeted DNA molecule.
What are the components of qPCR and what is their function?
- Buffer- Kcl2 to maintain pH.
- Taq Pol-an enzyme that synthesis DNA strands and adds nucleotides to the primers
- dNTP’s- facilitates the building blocks for DNA synthesis
- Syber green-binds to any dsDNA
- Primers-DNA sequences complimentary to the target region
- DNA template: contains sample DNA target sequence
- MgCl2- co factor required for Taq Pol
Explain what is meant by RPLA?
RPLA-reversed passive latex agglutination
The Ab attached to particles, reacts with the soluble Ag.
Latex particles do not partake in reaction.
Cross-linking of latex particles by the Ag/Ab reaction results in latex agglutination.
Explain the principle of RPLA?
Latex particles purified with antiserum taken from rabbits, immunised individually
with purified staphylococcal enterotoxins A, B, C and D.
Latex particles will agglutinate in the presence of the corresponding enterotoxin.
The test is performed in V-well microtitre plates.
Dilutions of the food extract or culture filtrate are made in five rows of wells, a volume of the appropriate latex suspension is added to each well and the contents mixed.
If staphylococcal enterotoxins A, B, C or D are present, agglutination occurs, which results in the formation of a lattice structure.
If staphylococcal enterotoxins are absent or at a concentration below the assay detection level (1ng/g food), no such lattice structure can be formed (tight button observed).
Explain LAMP?
Loop mediated isothermal amplification.
this technique is used for the amplification of a target gene.
List and describe the components for LAMP?
1) Betaine: helps amplify GC rich DNA.
2) Dntp’s: building block for DNA synthesis
3)Buffer: maintains pH.
4) Pol-Bst: used for strand displacement activity
5)4-6 primers: 2, inner, 2 outer, 2 loop
6) Syber green: optical analysis of LAMP product
