Principle

Materials/Apparatus
Steps of Procedure
Results and its interpretation
Uses

BY
RABIA
BS………….
Agenda of Discussion

 Biosafety rules for biology labs

 Biosafety levels 1, 2 and 3
 Sample collection procedure for bacteria and human blood
 Culturing of bacterial samples on differnt types of solid and liquid media
 Atleast 10 Biochemical tests for testing of one bacterium of your own choice
(Principle, procedure, observation and conclusion)
 DNA extraction from human blood, tissue and bacteria (Theory and
procedures (Organic and Inorganic + Any one kit method)
 Polymerase Chain Reaction (Principle, Procedure, Types and Applications
Biosafety rules for biology labs

Lab Safety Rules

 Lab Safety Rules 1-Wear protective clothing .
 Protective clothes o Gloves are essential. o Lab coats are required. o Safety
glasses ( goggles) may be required to avoid splashes.
 2-Laboratory personnel should not wear sandals Lab Safety Rules
 Lab Safety Rules Do NOT Wear: • Jewelry • Loose or Baggy clothing
 3-Avoid touching objects (e.g., pencils, cell phones, door handles) while wearing
gloves. Lab Safety Rules
 4-Pencils, labels, or any other materials should never be placed in your mouth.
Lab rules

 Long hair must be tied back or covered to minimize fire hazard or contamination of experiments. Lab Safety Rules
 Do not eat food or drink water in the lab. do not use lab glassware as food or water containers.
 Protect your hands safety, wash hands after every lab. - Handle glassware, sharp tools and heated containers carefully.
 keep all electrical cords and wires away from water .
 Never touch, taste or smell a chemical unless instructed to do so. - never mix chemicals unless instructed to do so.
-keep lids on chemical containers when not in use.
 Do not take any cultures out of the lab for any reason. • All cultures should be handled as potentially pathogenic. •
Liquid cultures must always be kept in a test tube rack.
 Do not engage in practical jokes or horseplay in the lab.
 Keep nonessential books and clothing far away from your work area.
 Wipe the bench tops down with disinfectant both before you begin your work and after you have completed your
work.
 Dispose of waste products according to instructions.
 Report all accidents, no matter how minor, to your supervisor!! Lab Safety Rules
Biosafety levels 1, 2 and 3

BSL(Biosafety level ) 1
 Frequent handwashing
 Door that can be kept closed when working
 Limits on access to the lab space when working
 No smoking,eating,drinking,storage of food in laboratory
 Care to minimize splashes and actions that may create aerosols
 Decontamination of work surfaces after every use after any spils
Biosafety level 2

 Agents associated with human disease

 Generally required for any human derived blood,bodily fluids,tissues in which
infectious agent may be unknown
 Agent include measles virus,Salmonella species,pathogenic
Toxoplasma,Clostridiumbotulinum,hepatitis B virus
Biosafety level 3 (BSL-3)

 Standard practices include BSL-2 PLUS:

 Strictly controlled access to the lab;
 Specific training for lab personnel in handling potentially lethal agents;
 Decontaminating all waste;
 Changing contaminated protective lab ,clothing,decontaminating lab clothing
before laundering;
 Insitutional policies regarding specimen collection and storage from workers to
establish exposure
Culturing of bacterial samples on differnt
types of solid and liquid media

 Solid media- contains 2 %agar

 Colony morphology,pigmentation,hemolysis can be appreciated.
 Eg :Nutrient agar,Blood agar

 Liquid media- no agar

 For inoculum preparation,Blood culture,continous

culture

Solid and liquid
Catalase Test

 This test is used to identify organisms that produce the enzyme, catalase. This
enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen
gas.
 The bubbles resulting from production of oxygen gas clearly indicate a catalase
positive result. The sample on the right below is catalase positive. The
Staphylococcus spp. and the Micrococcus spp. are catalase positive. The
Streptococcus and Enterococcus spp. are catalase negative.
Coagulase test

 Coagulase is an enzyme that clots blood plasma. This test is performed on Gram-
positive, catalase positive species to identify the coagulase positive
Staphylococcus aureus. Coagulase is a virulence factor of S. aureus. The
formation of clot around an infection caused by this bacteria likely protects it
from phagocytosis. This test differentiates Staphylococcus aureus from other
coagulase negative Staphylococcus species
Oxidase Test

 Principle:
 The oxidase test is designed for specifically detecting the presence of the terminal
enzyme system in aerobic respiration called cytochrome C oxidase or cytochrome a3.
Cytochrome C oxidase is the terminal or last H2 electron acceptor in aerobic respiratory
mechanism which is composed of a number of enzymes which alternatively oxidize and
reduce each other by donating or accepting electrons derived from H2.
 Procedure:
 Take a commercially available oxidase disc containing the reagent.
 Pick the isolated colony to be to be tested and rub in the disc.
 Observe for colour change within 10 seconds.
Indole Test

 Principle:
 Tryptophan is an amino acid that can undergo deamination and hydrolysis by bacteria that express tryptophanase
enzyme. Indole is generated by reductive deamination from tryptophan via the intermediate
molecule indolepyruvic acid. Tryptophanase catalyzes the deamination reaction, during which the amine (-
NH2) group of the tryptophan molecule is removed. Final products of the reaction are indole, pyruvic acid,
ammonium (NH4+) and energy. Pyridoxal phosphate is required as a coenzym.
 Procedure of Indole Test:
 Take a sterilized test tubes containing 4 ml of tryptophan broth.
 Inoculate the tube aseptically by taking the growth from 18 to 24 hrs culture.
 Incubate the tube at 37°C for 24-28 hours.
 Add 0.5 ml of Kovac’s reagent to the broth culture.
 Observe for the presence or absence of ring.
Result:
 A positive reaction is denoted by the appearance of a blue to blue-green color change on the bacterial smear within 2-3
minutes.
DNA extraction from human blood, tissue and
bacteria

 DNA extraction is a routine procedure to isolate & collect DNA.

 DNA extraction is the first step for subsequent molecular or forensic analysis.
 DNA from a single cell is not visible to the naked eye.
 However,when DNA is extracted from multiple cells,the amassed quantity can
easily be seen and looks like strands of mucous-like ,translucent cotton.
DNA extraction from bacteria

 When bacteria are lysed under alkaline conditions both DNA and
 Proteins are precipitated.After the addition of acetate-containing
 Neutralization buffer the large and less supercoiled chromosomal DNA and
proteins participate,but the small bacterial DNA plasmids can renature and stay in
solution.
Principle of PCR

 PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from
deoxynucleotide substrates on a single-stranded DNA template. DNA polymerase
adds nucleotides to the 3` end of a custom-designed oligonucleotide when it is
annealed to a longer template DNA. Thus, if a synthetic oligonucleotide is
annealed to a single-stranded template that contains a region complementary to
the oligonucleotide, DNA polymerase can use the oligonucleotide as a primer and
elongate its 3` end to generate an extended region of double stranded DNA.
Procedure:

 1. Denaturation

 The DNA template is heated to 94° C. This breaks the weak hydrogen bonds that hold DNA strands together in a helix, allowing the
strands to separate creating single stranded DNA.

 2. Annealing

 The mixture is cooled to anywhere from 50-70° C. This allows the primers to bind (anneal) to their complementary sequence in the
template DNA.

 3. Extension

 The reaction is then heated to 72° C, the optimal temperature for DNA polymerase to act. DNA polymerase extends the primers, adding
nucleotides onto the primer in a sequential manner, using the target DNA as a template.

 With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These
Types of PCR

 Real-time PCR
 Quantitative real time PCR (Q-RT PCR)
 Reverse Transcriptase PCR (RT-PCR)
 Multiplex PCR
 Nested PCR
 Long-range PCR
 Single-cell PCR
 Fast-cycling PCR
 Methylation-specific PCR (MSP)
 Hot start PCR
 High-fidelity PCR
 In situ PCR
Applications of PCR

 PCR is used in analyzing clinical specimens for the presence of infectious agents, including
HIV, hepatitis, malaria, anthrax, etc.
 PCR can provide information on a patient’s prognosis, and predict response or resistance to
therapy. Many cancers are characterized by small mutations in certain genes, and this is what
PCR is employed to identify.
 PCR is used in the analysis of mutations that occur in many genetic diseases (e.g. cystic
fibrosis, sickle cell anaemia, phenylketonuria, muscular dystrophy).
 PCR is also used in forensics laboratories and is especially useful because only a tiny amount
of original DNA is required, for example, sufficient DNA can be obtained from a droplet of
blood or a single hair.
 PCR is an essential technique in cloning procedure which allows generation of large amounts
of pure DNA from tiny amount of template strand and further study of a particular gene.