Micro prac Flashcards
Describe how virus neutralisation tests work
POSITIVE SAMPLE - anti-viral Ab’s present, add viral Ab’s to cultured cells -> anti-viral Ab’s inhibit viral attachment and replication, no damage to cells
NEGATIVE SAMPLE
anti-viral Ab’s absent, add viral Ab’s to cultured cells where the virus can replication -> causes visible damage
What are 3 features of immunohistochemistry
- Labelled antibody
- Binds to antigen
- Localises site of replication or expression within the tissue
What are the 3 steps of PCR
- Melting: denaturing of the DNA duplex at a high temperature to yield single stranded DNA.
- Annealing: primers anneal to the single stranded target DNA sequence.
- Elongation: DNA polymerase extends the primers by adding dNTPs to the phosphate backbone.
What are 4 steps in quantitative (real time PCR)
Uses fluorescent dyes or probes to detect DNA amplification
Detect fluorescence after each cycle
Fluorescence exceeds background threshold
Allows quantification of target in original sample
Microsporum canis what is it, reservoir species, macroculture feature, clinical significance and what agar used to grow it
A fungal zoophilic dermatophyte
Reservoir -> cats, dogs, horses, humans
Macroculture -> colonies are flat, spreading, white to cream coloured, bright golden yellow to brownish yellow
Clinical significance -> ringworm in humans and skin lesions in animals
Agar -> Malt Extract Agar
Aspergillus Fumigatus what is it, reservoir species, macroculture features, clinical significance and what agar used to grow
A fungus
Reservoir -> almost everywhere on every substrate
Macroculture -> A. fumigatus -> grey-green
Clinical significance -> most common cause of invasive and non-invasive aspergillosis
Agar -> Sabouraud’s agar
Virus what need to grow in and what results in
Require Living cells (cell or tissue cultures)
Generally cause cytopathic effects (CPE) -> cells look sick
Equine Herpes Virus-1 what is the most common cells cultured within and what the cells look like in an uninfected and infected culture
Kidney cells derived from equine foetuses (EFK)
Uninfected culture -> typical mosaic-like pattern (EFK), cells grown as a monolayer
Infected culture -> virus “plaque” formed by virus growth leading to lysis of the infected cell
- the foci of the infection slowly spreads or enlarge until the entire cell culture is wiped out
Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus agalactiae what agar grown on (characteristics, what used for) and what look like in a culture
Grown on Sheep Blood Agar -> non selective medium and therefore allows the growth of a wide variety of organisms, detect sample purity, colony morphology and haemolysis
Pseudomonas aeruginosa -> blue/green pigment and sweet odour
Staphylococcus aureus -> smaller colonies compared to psudomonas
Streptococcus agalactiae -> are small colonies that are often beta-haemolytic (complete lysis of sheep blod cells in the vicinity of the colonies)
Pseudomonas aeruginosa and Staphylococcus aureus what can also be grown on (characteristics, what used for) and what can’t grow on it
Nutrient agar -> non-selective medium, less nutritious than blood agar, doesn’t support the growth of the same variety of organisms
-> easier to see the blue/green pigment of pseudomonas aeruginosa
Strep. Agalactiae isn’t supported by nutrient agar
E. coli and Salmonella typhimurium what agar grown on (characteristics and what grown on that agar), and the differences in look
MacConkey agar -> used to isolate and enumerate coliforms (Gram negative organisms of GIT of mammals)
Selective Differential Medium -> selective ingredient bile salts -> inhibit organisms that do not tolerate the presence of bile
Organisms that grow on that agar differentiated by their effect on neutral red (pH indicator) indicator of lactose fermentation
E. coli -> Yes lactose fermentation - acid reaction PINK COLONIES
Salmonella -> no lactose fermentation - alkali reaction, TRANSPARENT/PALE COLONIES
Salmonella what is the other agar that it can be grown on other than MacConkey agar and what does it look like
XLD agar -> used for the isolation and presumptive indentification of salmonella
On XLD agar salmonella colonies are black, indicative of H2S production
Methicillin resistant Staphylococcus aureus (MRSA) what agar is it grown on (characteristics and features, what grow) and what does it look like
Mannitol salt agar (MSA) is a selective-differential medium
- high salt concentration selects organisms that tolerant these conditions (surface of the skin), differentiated by their ability to utilise mannitol and detected by inclusion of pH indicator
MRSA - colonies appear opaque and are surrounded by a yellow halo
MRSA II culture can also be used -> typical colonies look rose and mauve in colour
-> antimicrobial susceptibility plate tests for the resistance and susceptibility of the isolate to range of antimicrobial agents -> growth within annular radius of less than 6mm indicates resistance
List the steps of the gram stain and the result
1) Apply crystal violet
2) leave for 1 minute -> Rinse
3) Apply Gram’s iodine
4) leave for 1 minute -> rinse
5) Acetone/alcohol -> rinse
6) apply Dilute Carbol fuchsin
7) leave for 1 minute
8) Rinse and blot dry
Gram positive -> PURPLE -> thick peptidoglycan retain satin
Gram negative -> PINK -> thin peptidoglycan, stain is lost
Using the microscope to look at bacteria, what are the 2 different conditions, objective lens, oil or non-oil, condenser up or down
1) Higher magnification ie gram smear
- 100x objective lens
- immersion oil
- condenser up
2) Lower magnification -> not for fine observations of bacteria
- x4, x10 and x40 objective lenses
- no immersion oil
- condenser down
What is the best way to test for EHV4 and EHV1 viruses and how use
Indirect ELISA
1) determining the epidemiology of EHV4 and EHV1
2) Identify horses that are latently infected carriers of EHV1 so managed on stud farms
3) Determine the cause of abortion where no foetal tissue samples can be obtained -> acute and convalescent serum
EHV4 and EHV1 what family of viruses, features and what cause
EHV4 -> Rhinopneumonitis
- respiratory disease in young horses
- treat with vaccine
EHV1 -> most important cause of viral abortion in mares can also lead to respiratory disease in foals, endemic in populations,
- can lead to abortion storms
1. respiratory spread from other horse 2. reactivation of latent virus -> keep mares separated
Aborting mare is infectious for 1-2 days from reporudctive tract (if infecting stallion) or 2 weeks from respiratory tract
Alphaherpesvirinae -> enveloped dsDNA, inactived by heat and pH, lifelong latent infection -> continours or periodic shedding
List the 6 steps of an indirect ELISA
1) Coat well with specific antigen (virus)
2) Block unused sites with unrelated Ag
3) Add test sera (Primary antibody present - Ab)
4) add enzyme-conjugated secondary Ab directed against Primary Ab
5) Add substrate that results in colour presentation of the secondary-primary Ab-antigen complex
6) read absorbance
<0.1 negative
0.1-0.2 - indeterminate (repeat test)
>0.2 positive
Acute (a) and convalescent (c) serum what are the 4 scenarios with indirect ELISAs
1) Absorbance below 0.2 -> negative with no change between a and c
2) If above 0.2 in acute and increasing in con then been exposed recently
3) If -ve and then +ve then again exposed recently
4) If +ve and decreasing or the same in con then not exposed recently
+ve >0.2
Direct ELISA List the steps
1) Coat wells with specific Ag (virus)
2) Block unused sites with unrelated Ag
3) Antibody conjugated with enzyme is added and incubated with the antigen
4) Add substrate that results in colour presentation of the secondary-primary Ab-antigen complex
6) read absorbance
<0.1 negative
0.1-0.2 - indeterminate (repeat test)
>0.2 positive
Sandwich ELISA what type of ELISA and the features
- capture antibody is used first and sticks to the ELISA plate
DIRECT - detection antibody is conjugated to an enzyme
INDIRECT - detection antibody is unlabeled and the secondary detection antibody is conjugated to an enzyme
How to graph results for ELISA and what occurs with the plateau and how to tell if animal infected recently
Absorbance (directly related to concentration of antibodies) VS Dilution of the plasma samples
Plateau -> saturation of the antigen by antibodies, this point depends on the assay
THEREFORE if doing calculations needs to be on the non-plateau areas
If infected recently then look at amount of absorbance if 4 fold increase between acute and convalescent of IgG concentration -> recent immune response
Streptococcus agalactiae, Staphylococcus aureus and Pseudomonas aeruginosa, clostridium gram positive or gram negative and shape
Staphylococcus aureus -> gram positive cocci in chains or diplococci
Streptococcus agalactiae -> gram positive cocci in clusters
Pseudomonas aeruginosa -> gram negative bacilli
Clostridium -> gram positive bacilli
Agglutination tests, when does it occur, when used and advantage and disadvantage
When Ab combines with its specific antigenic determinant or epitope on the surface of particulate Ag, agglutination occurs
Blood typing, pleuropneumonia of cattle, pullorum disease in chickens
Advantage -> animal can be held until results are obtained
Dis -> occurrence of significant number of false negatives and false positives
Haemagglutination assay what does it measure and the 3 scenarios
Measurement of antibody
1) No antibodies present -> so cells settle to the bottom -> red dot
2) Antibodies present -> Antibody binds to the RBCs creating a raft (murky)
3) Too many RBCs for the amount of antibodies present -> some of the RBCs settle to the bottom (red dot)