1. Diagnostic Methods in Microbiology lab.
OPT 435
Identifying the organism causing an infectious diseases is important for effective treatment

2. Diagnostic Microbiology
Means of laboratory diagnosis of microorganisms.
Definitive microbiologic diagnosis of an infectious disease usually involves one or more of the following five basic laboratory techniques.
Direct microscopic visualization of the organism.
Cultivation and identification of the organism.
Detection of microbial antigens.
Detection of microbial DNA or RNA.
Detection of an inflammatory or host immune response to the microorganism.

4. Microbial Genetics Genes: units of heredity
Genetics: Study of heredity
Heredity: Passing of genetic information from an organism to it’s offspring
Genes: units of heredity
Determines traits/ characteristics an offspring will have.
Located on chromosomes.
For each trait-> minimum of 2 genes. One from mom, one from dad.
There are many genes located on a Chromosome.

5. DNA, Genes and Chromosomes
台大農藝系 遺傳學
DNA, Genes and Chromosomes
Genetic material of both eukaryotes and prokaryotes is DNA (deoxyribonucleic acid). Many viruses also have DNA, but some have RNA genomes instead.
2.DNA has two chains, each made of nucleotides composed of a deoxyribose sugar, a phosphate group and a base. The chains form a double helix

6. DNA, Genes and Chromosomes
台大農藝系 遺傳學
DNA, Genes and Chromosomes
3.There are four bases in DNA:
A (adenine), G (guanine), C (cytosine) and T (thymine).
In RNA, U (uracil) replaces T.
b. The sequence of bases determines the genetic information.
c. Genes are specific sequences of nucleotides that pass traits from parents to offspring.

7. DNA, Genes and Chromosomes
台大農藝系 遺傳學
DNA, Genes and Chromosomes
Genetic material in cells is organized into chromosomes (literally “colored body” because it stains with biological dyes).
a. Prokaryotes generally have one circular chromosome.
b. Eukaryotes generally have:
i. Linear chromosomes in their nuclei, with different species having different numbers of chromosomes.
ii. DNA in organelles (e.g., mitochondria and chloroplasts) that is usually a circular molecule.

8. Structure of DNA
Long chain of repeating units ( polymer) called nucleotides.
A nucleotide unit contains: 1. phosphate group O 2. deoxyribose (sugar) 3. nitrogenous base:
A- adenine
T- thymine
C- cytosine
G- guanine

9. Structure of DNA

10. One Strand of DNA
The backbone of the molecule is alternating phosphate and deoxyribose, a sugar, parts.
The teeth are nitrogenous bases.
phosphate
deoxyribose
{Point to the 3-D mode, if you have one, to show the parts as you discuss them.}
bases

11. Two Stranded DNA
Remember, DNA has two strands that fit together something like a zipper.
{Point to the 3-D model to show the parts as you discuss them.}

12. Structure of a Double Helix
Sides of the “ladder” are alternating phosphate group and deoxyribose sugar.
“rungs” of the ladder are made of 2 nitrogenous bases.
Specific pairings:
There is a weak Hydrogen bond Between the base pairs.
Structure as a double helix
When a cell goes through mitosis(cell division) the DNA must also make a copy of itself.

13. How does DNA replicate?

15. PCR Technique
The Polymerase Chain Reaction (PCR) is a technique which amplifies a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence
-Amplify= making numerous copies of a segment of DNA
It is a revolutionary method developed by Kary Mullis in the 1980s
As the name implies, it is a chain reaction: one DNA molecule is used to produce two copies, then four, then eight and so forth.

16. PCR Technique
This continuous doubling is accomplished by specific proteins known as polymerases, enzymes that are able to string together individual DNA building blocks to form long molecular strands.
To do their job polymerases require a supply of DNA building blocks, i.e. the nucleotides consisting of the four bases adenine (A), thymine (T), cytosine (C) and guanine (G).
They also need a small fragment of DNA, known as the primer, to which they attach the building blocks as well as a longer DNA molecule to serve as a template for constructing the new strand

17. PCR Technique
If these three ingredients are supplied, the enzymes will construct exact copies of the templates.
PCR is a method used to acquire many copies of any particular strand of nucleic acids
In this method we amplify the DNA of the microorganism.
Rapid, the genetic material of the microorganism will be amplified billion times within hours.
More sensitive and specific than conventional techniques.
Can suggest the diagnosis in the presence of even a few organisms in the sample.

18. Three major steps involved in the PCR technique
Step 1 ( denaturation 95°C):
the target DNA containing the sequence to be amplified is heat denatured to separate its complementary strands.
Step2 (annealing 50°C):
the temperature is lowered so that the primers can anneal ( attach) to the complimentary DNA
Step3 (extension 70°C):
Taq DNA polymerase extends the primers and synthesizes copies of the target DNA sequence by adding the corrosponding nucleotides.

19. What you need for PCR
Two short DNA fragment that stick specifically to each
of the DNA strands at some distance of each other
Primers
Can be specific for:
A certain bacterium
Bacterial species
Genes (e.g., toxin gene)

20. What you need for PCR Apparatus to perform about 35 cycles of a three
temperature procedure
95 °C (denaturation of DNA)
50-60 °C (annealing of primers)
72 °C (extension of the primers)

21. What you need for PCR Put into one reaction tube: PCR tube
Sample (+/- target DNA)
Primers for the specific detection
Nucleotides
Enzyme
PCR tube
THERMOCYCLER

22. Performing PCR Put your tube in the apparatus
Let the program run (35 cycles)
If primers fit, there is amplification of target DNA
If primers do not fit, no amplification product
=> the DNA (micro-organism) was not in the sample
Detect if there is PCR product

23. Detection and analysis of PCR products
To check whether the PCR generated the anticipated DNA fragment agarose gel electrophoresis is employed for size separation of the PCR products.
The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products
Ethidium bromide is a fluorescent dye that intercalates into the DNA. Size markers can be electrophoresed on the gel to allow size determination of the PCR product.

24. PCR technique application Examples
Detection of pathogens: used to identify (bacteria, viruses, fungi, or parasites).
Viruses
HIV, SARS, H5N1, CMV, HSV, HZV.
Bacteria
meningococcus, legionellosis
Analysis for resistant genes
MRSA, VRE
Classification of organisms
Mutation detection
DNA fingerprinting
Genetic engineering
Pre-natal diagnosis
Conduct forensic investigations
Gene therapy

25. Real-time PCR
Also called quantitative real time polymerase chain reaction is a laboratory technique based on the PCR
the same principle of amplification is employed in real- time PCR. But instead of looking at bands on a gel at the end of the reaction, the process is monitored in “real- time”
Real-Time PCR permits the analysis of the products while the reaction is actually in progress. This is achieved by using various fluorescent dyes which react with the amplified product and can be measured by an instrument. This also facilitates the quantitation of the DNA.

26. Real-time PCR

27. Advantage over normal PCR
Real Time PCR has many advantages over normal PCR:
It does not require gel preparation like traditional PCR.
It is not time consuming like normal PCR.
No contamination
quantification of sample.

28. Multiplex PCR
Multiplex PCR is a molecular biology technique for amplification of multiple targets in a single PCR experiment.
In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has the potential to produce considerable savings in time and effort within the laboratory without compromising on the utility of the experiment.

29. Introduction to Ab-Ag Binding
Antigens: any agent (microorganism, molecule, protein…etc) that can stimulate the production of antibodies.
Antibodies Specific glycoprotiens produced by lymphocytes in response to the presence of an antigen.
All antibodies are in a class of proteins called Immunoglobulins.
Each antibody is specific to the
antigen that stimulates its production.

30. Introduction to Ab-Ag Binding
The Ag & the Ab combine specifically with each other. This interaction between them is called Ag-Ab reaction

31. Introduction to Ab-Ag Binding
These reactions form the basis for detection of infectious disease causing agents and also some non-specific Ag’s like enzymes.
When Ag-Ab reactions occur invitro, they are known as serological reactions.
Serology is the part of the laboratory that detect the production of Ab in patient serum to infection.
Immunologic methods are any laboratory method that use the Ag-Ab binding (reaction) to diagnosis a disease.

32. Detection of Microbial Ag
These methods depend on detection of microbial antigen with known antiserum.
These methods of identification are often rapid.
Example of these tests:
Immunofluorascent assay (IFA).
Enzyme-Linked Immunosorbent Assay (ELIZA)

33. Immunofluorescent Assay (IFA)
Serum & fixed organisms are combined.
If Ab against this organisms Ag’s is present in Patient Serum they will form Ag-Ab complex.

34. Immunofluorescent Assay (IFA)
An Ab that is labeled with fluorescence is added & it bind to the Ag-Ab complexes.
The fluorescent tagged Ag-Ab complexes are examined with a fluorescent microscope.

35. Immunofluorescent Assay (IFA)

36. Test Done in the Microbiology Lab
Immunofluorescence staining:
Usually for viruses.
HSV-1 , virus infected cells showing apple green fluorescence.

37. Test Done in the Microbiology Lab
ELISA (Enzyme-Linked Immunosorbent Assay):
Immunoassays come in many different formats and variations.
There are two main variations on this method:
The ELISA can be used to detect the presence of antigens that are recognized by an antibody or it can be used to test for antibodies that recognize an antigen.

38. Enzyme-Linked Immunosorbent Assay (ELISA)
The enzyme-linked Ab reacts with the Ag-Ab complexes , activating the enzyme.
The enzyme activity (color change) is then quantified.

39. ELISA A general ELISA is a five-step procedure:
1) coat the microtiter plate wells with antigen;
2) block all unbound sites to prevent false positive results; 3) add primary antibody (e.g. rabbit monoclonal antibody) to the wells;
4) add secondary antibody conjugated to an enzyme (e.g. anti-mouse IgG)
5) reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction.

40. ELISA

41. ELISA Patient Serum Ab that is not specific to the Ag
Ab that is specific to the Ag

42. ELISA
Wash out non-specific Ab

43. Ab labeled with an enzyme
ELISA
Add the Ab- that is labeled with an enzyme
Enzyme
Ab labeled with an enzyme

44. Ab labeled with an enzyme
ELISA
Add the substrate that will react with the enzyme and give a color reaction, we measure this color and it is an indication of the presence of the specific Ab in patient serum.
Substrate
Enzyme
Ab labeled with an enzyme

46. Example of ELISA application
Toxoplasmosis:
used for the detection of toxoplasmosis.
Keep in mind that peak of Ab against Toxoplasmagondii is at 6 – 8 weeks and this peak gradually decline over months –years.
In the food industry in detecting potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs
used in toxicology as a rapid presumptive screen for certain classes of drugs
The other uses of ELISA include:
detection of Mycobacterium antibodies in tuberculosis
detection of hepatitis B markers in serum
detection of enterotoxin of E. coli in feces
detection of HIV antibodies in blood samples
Detection of Chlamydia.