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Flashcards in Lecture 23 Deck (36):
1

Procedure of Isolation of Bacterium

Streaked onto agar plate
Grown in general purpose media
Enrichment culture to isolate specific microbes
Agar plates are incubated in appropiate temperatures

2

Reading

Organisms of interest are sub-cultred by streak plating onto differential agar or TSA

3

Confirmation

Pure culture obtain, classical identification, biochemically adn antimicrobial susceptibility testing.

4

Identifying pure bacteria cultures

Colony morphology, Colour, Shape, Margins , elevation

5

Diagnostic Test - Catalase

h2O2 , postive reaction shows gas formation. Differentiates negative Streptococcus from postivie Stahpylococcus

6

BIochemical Tests

Physiologcila reactions to nutrients as evidence of absense or presense of enzymes

7

Carbohydrate fermentation

Differential medium shows fermentation 9acid production) and gas formation
Acid - yello
Acid nd gas - yellow and gas
No acid - stays red
Use durham tube

8

Methyl Red test

Acid production test - bacteria grown in MR -VP broth
Several drops of methyl red
Positive - Red
Negative - Yellow - Orange

9

Nitrate Reduction

After 24-48 hours Nitrate reagents are added.
Red - Nitrate has been reduced to nitrate

10

Starch Hydrolysis

Iodine solution is added,
Positive - colourless area around growth
Negative
no growth

11

API 20 Strips

Rapid Test where the identification of Enterobacteriae and other gram negative bacteria can be established

12

API 20 Strips

5 to overnight hours the 20 tests are converted to 7 - 9 digital profiles

13

API 50 Strips

Carbohydrates , identification of lactobacilli, bacilli and enteric bacteria

14

BBL Crystal Enteric / Nonfermenter ID Kit

Modified microplate with 30 wells of organic and inorganic substances for identification of the Enterobacteriacecae and other gram negative bacilli

15

Antimicrobial Testing

1. Disc Diffusion Method,, lawn of pure bacterial culture, discs added therefore show ZOI
2. Broth Dilution Method, well with lowest concentration of antibiotic that shows no visible growth , highest concentration of antibiotic is Left

16

Etest Method

Strip test for antibiotic suseptibility
Each strip is calibrated in terms of the minimum inhibitory concentration MIC
the concentration is the lowest at the centre of the plate
Lowest concentration of antibiotic that inhibts bacterial growth is the MIC value for that particular agent

17

Molecular Techniques can be used to identify or characterise an isolated organism

Genes can be used to infer relationships between organisms,
genes can be ribosomal, houskeeping, virulence
Genes perferables conserved nd showing a small rate of change overtime 16S rRNA

18

DNA Diagnostic Systems

DNA Sequencing , Nucleioc acid probes, PCR, Restiction endonuclease analysis , Random amplified polymorphic DNA , DNA fingerprinting

19

Characteristics of a Good detection system

Sensitivity, Specificity , Simplicity

20

Sensitivity

Detect very small amounts of target even in the presense of other molecules

21

Specificity

Test yields a postive result for the target molecule

22

Simplicity

Test runs efficiently and inexpensively

23

Nucleic Acid Probes

Nucleic Acid Hybridization most powerful tools avalible for microbe identification
Detects specific DNA sequences
Nucleic Acid probe specific for that organism

24

Nucleic Acid Probe

A short specific sequence of DNA that is used to query whether a sample contains target DNA or DNA complementary to the gene probe

25

Membrane Filter Assay

Colonies from plates or samples of infected tissues are treated with a strong alkali lysing the cells to generate a single stranded DNA. then fixed to a filter.
Hybridizatoin is carried out by incubating at a temperature neccesary to form a stable duplex between the target and probe. Following wash, the extent of hybridization can be measured

26

Dipstick Assay

Dual reporter and capture probes are used
Capture probe contains a poly(dA) tail that hybridises to a poly(dT) oligonuelciotide affixed to the dipstick, binding to the oligonucleotide-target -report complex

27

Advantages of Nucleioc Acid Probes

More stable at high temperatures, pH , presense of organic solvents
Specimen can be treated harshly
Can identify micoorganisms that are no longer alive
Are more specific than antibodies

28

Detection of Malaria

Blood smears , ELISA(do not differentiate between past and present infection)
A DNA diagonistic system would only measure current infection

29

Detection of Malaria procedure

Genomic library of the parasite screened with probes
Probes then hybridised strongly with repetitive DNA
Probes were tested for ability to hybridise to other plasmodium species which do not cause malaria, and to human DNA
Probes that hybrizided to P. falciparum only could be used as a diagnostic tool

30

PCR

many cases there is not enough DNA in a sample for a gene probe to detect , therefore DNA is amplified using PCR

31

PCR

Uses two sequence specific oligonucleotide primer to amplify the target DNA Reverse and Foawrd
Presence of the approopriate amplified size fragment confirms the target is present

32

Primers for the following pathogens are now available

E.coli, M. tuberculosis, HIV

33

RNA to be amplified

Use reverse transcription to CDNA

34

PCR product confirmation

Correct product size, Sequence the product, hybridisation - use a gene probe to confirm product, used nested or semi nested PCR

35

nested PCR

Uses two sets of primer
After amplification, first set of primers is a second set of oliognucleotides complementary to amplified sequence is added
Nested PCR increases the sensitivity of the assay

36

Quantitative Real Time PCR ....

One to count numbers of gene in a sample
Quantitation of DNA and RNA is possib