Definition: The branch of biology that deals with the study of microorganism and their effects on other living things. Introduction: In microbiology different microorganism are studied like bacteria, viruses, fungi and protozoa. This discipline also helps us to understanding the other aspects like physiology, biochemistry, ecology, cell biology and clinical findings of microorganisms. For clinical diagnosis different culture tests are used to identify the presence of type of microorganism.
Following are the cultures which are being used; • Blood culture and sensitivity • Puss culture • Urine culture • Body fluid culture These culture tests are performed on specific culture media. Following are the different types of culture media; • MacConkey agar • CLED agar for urine • Mannitol salt agar • Blood agar Blood for culture and Sensitivity Introduction: It is a culture test through which blood culture is done. If microbe’s colony is found on culture, it means that an infection is present, then sensitivity can be applied according to the requirement. Through this culture, type of microbes can be identified.
Principle: Several types of microbes may be present in blood which causes the hemolysis of RBC, s and hemoglobin by secreting enzymes like hemolysins. These bacteria may be Gram- positive or Gram-negative cocci. Through this culture, type of bacteria is identified. Composition of Blood Agar: • 0.5% NaCl • 1.5% agar • 5% Sheep Blood • Buffer (pH 7.2-7.6) • 0.3% yeast extract • Distilled water • 0.5% Peptones Preparation: • In one litter of distilled water mix 28g of nutrient agar.
• By heating this mixture stirring continuedly by stirrer to mix all components equally. • For 15 minutes autoclave to this mixture at 121 Celsius. • Allow it to cool at 45-50 degree but nit solidify. • Add 5% sterile defibrinated blood.
• Avoid air bubbles. • Distribute it into the petri dishes. Material: • Petri dishes. • Incubator. • Autoclave.
• Blood sample. • Glass slides. • Microscope. • Inoculating loop.
• Blood culture vials. • Stains. • Burner. Procedure: • Blood sample is collected into blood culture vials. • Incubate the petri dishes at 37 C before 24 hours of use. • Pour the already prepared blood agar into the petri dishes.
• Wait until this media become semi solidify. • Take a drop of blood and pour it on that blood agar media. • With the help of hot red inoculating loop, striking is done in zig zag manner. • Again, incubate these petri dishes at 37 C for 24 hours. • Observe the culture after 24 hours. • If the growth appears, go for the further process like preparing slides and staining.
• If growth is not found, go for the subculture. Results: Following three types of hemolysis may be present; • Alpha hemolysis: causes the partial destruction of RBC, s and greenish color of agar is appear around the colonies. • Beta hemolysis: causes the complete destruction of hemoglobin and RBC, s and clear medium around colonies is appears. • Gemma hemolysis: it is non-hemolysis and simple growth appears.
Urine for culture and sensitivity Introduction: To diagnose the diseases of urinary tract, urine culture test is done to identify the microbes causing urinary tract infections (UTI, s). For this culture test cysteine-lactose-electrolyte-deficient (CLED) agar is used, because this culture media supports the growth of those pathogens which causes the UTI, s like micrococci, lactobacilli etc. Composition of CLED agar: • Agar 15g/L • Bromothymol blue 20mg/L • Tryptone 4g/L • Peptone 4g/L • Lactose 10g/L • L-cysteine 128g/L • Lab lemco powder 3g/L Preparation: • Put the 36 g medium into one litter of distilled water. • Mix by heating while continues stirring with stirrer.
• Autoclave for 15 minutes at 121 C. • Pour into petri dishes. • Allow to semi solidify. Material: • Urine sample. • Petri dishes.
• CLED agar media. • Microscope. • Glass slides. • Stains and dyes. • Burner • Inoculating loop. • Autoclave.
• Incubator Procedure: • Collect urine sample. • Incubate the petri dishes at 37 C for 24 hours before use. • Take a drop of urine sample and spread it on the media by striking. • Incubate that media plates for 24 hours. • After 24 hours, observe the culture.
• If growth is present, go for further process like slide making and staining. • If growth is not present, go for subculture. Results: Sr.no Name of Bacteria Appearance 1 Klebsiella Much mucoid, varying color from yellow to white blue. 2 Salmonella Blue in color and flat colonies. 3 Corynebacterial Gray and small colonies. 4 Staphylococcus aureus Deep yellow and uniform in color, 0.75 mm in diameter.
5 E. coli Yellow in color, 1.75 mm in diameter 6 Proteus species Smaller than E. coli in size, translucent in color. 7 Pseudomonas aeruginosa Green colony in color, rough periphery. 8 Enterococcus faecalis Yellow in color, 0.5 mm in size Pus for culture and sensitivity Introduction: Pus is produced in wound when the wound is infected with bacteria. Pus culture test is done to identify the type of bacteria which causes the infection.
For this test MacConkey agar is used to culture the pus. MacConkey agar is selective and differential culture medium used to culture Gram-negative bacteria. Composition of agar: Following is the composition; No. Component name Quantity 1 Distilled water 1L 2 Agar 13.5g/L 3 Neutral red 0.03g/L 4 Crystal violet 0.001g/L 5 Proteose peptones 3g/L 6 Bile salt 1.5g/L 7 Sodium chloride 5g/L 8 Peptone 17g/L 9 Lactose 10g/L Preparation of MacConkey agar: Following steps are involve in preparation of MacConkey agar; • Pour 49.54 g of all ingredients in 1000 ml of distilled water. • Dissolve the medium by heating. • Autoclave for 15 minutes at 121°C and 15 lbs. pressure.
• Cool to 45-50°C. • Mix well to medium. • Pour in sterile petri dish. Required materials: Following materials are require; • Pus sample • Pus sample tubes • MacConkey agar • Petri dishes • Burner • Glass slides • Microscope • Stains and dyes • Inoculating loop • Autoclave machine Procedure: Following steps are involve; • Draw pus sample in pus sample tube. • Label it properly. • Incubate to petri dishes at 37°C.
• Pour pus sample on petri dishes. • Incubate the plate. • Observe the culture after 24 hours. • Record the observations.
Results: Following results may be; Sr.no. Organism Color Appearance of Colony 1 Aerobacter aerogens Pink Mucoid 2 Escherichia coli Red/pink Non-mucoid 3 Pseudomonas aeruginosa Green-Brown Fluorescent growth 4 Staphylococcus species Pale Pink Opaque 5 Enterococcus species Red Minute, Round Fluid complete examination Introduction: In laboratory, many types of fluids can be tested other than blood because these fluids tests can give more precise information about any part of the body. These fluids may be paracardial, Extracellular fluid, cerebrospinal fluid, peritoneal fluid, and pleural fluid etc. Principle: Fluid examination may be of the three types; • Physical Examination: Volume, Color, turbidity, coagulum, deposits. • Chemical Examination: Glucose, total protein, albumin, LDH. • Microscopic Examination: RBCS, WBCs, total cells, lymphocytes, polycytes, AFBs, morphology of cells, gram positive or negative bacteria.
Microscopy is done with Neubauer chamber (hemocytometer) or stained slides. Required materials: Following materials are required; • Cerebrospinal fluid sample. • Neubauer chamber. • Cover slip • Microscope. Procedure: Following steps are involve; • Make the dilution of blood sample.
• Place cover slip over the chamber. • The tip of the pipette is placed near the cover slip. • Capillary action will draw the fluid into the chamber • It is important not to overload the chamber, as doing so will give an inaccurate count. • Count the cells by applying formula; Total cells = No. of cells x 2.5 x dilution facto Staining: It is a technique involve in artificial coloring of a microorganism, cell or tissue to facilitate in differentiation and classification.
Stain: A penetrative dye which is used to coloring a microorganism, cell or tissue to facilitate in differentiation and classification. Types of staining: Following are the major types of staining; • Gram staining. • Giemsa staining. • Ziehl-Neelsen staining. Gram staining Introduction: Gram staining method originally devised in 1882 by Christian Gram.
We can differentiate between gram- positive and gram-negative bacteria through this type of stain. Gram-negative bacteria stain red color while gram-positive bacteria stain purple brown. Principle: When the bacterial smear is stained with primary stain (Crystal Violet) and fixed by the iodine, some of the bacteria retain the primary stain and some are decolorized by alcohol. Gram positive bacteria have low peptidoglycan and lipid content due to which cell shrinkage occur and pores of the cell membrane also closed, and crystal violet stain is not removed.
This causes the purple and blue appearance of gram positive bacteria. Gram negative bacteria have high content of peptidoglycan and lipids. So, gram negative bacteria stain red due to safranin. Required material: Following materials are required; • Primary stain (crystal violet) • Mordant (iodine) • Decolorizer(methanol) • Counterstain (safranin) • Clean glass slide • Microscope • Inoculating loop • Burner • Bacterial culture Procedure: Following steps are involved; • Take a grease free slide.
• Prepare a smear on the clean slide. • Air dry. • Heat fix • Crystal Violet was poured. • Wait for 30 seconds to 1 minutes.
• Rinse with tap water. • Flood iodine for 1 minute. • Wash with tap water. • Wash with 95% alcohol for about 10-20 seconds.
• Rinse with tap water. • pour safranin for about 1 minute. • Wash with tap water. • Air dry. • Blot dry. • Observe under Microscope.
Interpretation: Gram positive bacteria like Clostridium, Actinomyces, Staphylococcus, Streptococcus etc. stained purple blue color while gram negative bacteria like Escherichia coli, Helicobacter, Salmonella, Pseudomonas etc. are stained red which is shown in figure. Giemsa staining Introduction: Giemsa staining is commonly used for parasitic examination of blood. This type of staining also used for the examination of blood cells by Giemsa staining of blood smear.
Nuclear differentiation and morphological characteristics can be studied under the microscope by Giemsa stain. Principle: Giemsa stain contains both the Acidic and Basic dyes like the affinity of blood cells. The acidic dye like Eosin and Azure stains the basic components of the cells like the cytoplasm etc. Basic dye e.g. Methylene blue stains the acidic components, especially to cell nucleus. Required materials: Following materials are required; • Clean slide • Distilled water • Inoculating loop • Burner • Stock solution (Giemsa stain).
• Microscope Procedure: Following steps are involve; • Take a grease free slide. • Prepare a smear on the clean slide. • Air dry. • Heat fix • Dissolve 1ml of Giemsa stain into 4ml of distilled water. • Pour this dilution on smear which is prepared on the glass slide.
• Wait for 15 minutes. • Wash with tap water. • Observe under the microscope. Interpretations: Following are the interpretations; Parts of Cell Color • Nucleus • Blue • Basophilic cytoplasm • Blue • Neutrophilic granules • Lilac • Eosinophilic granules • Orange • Mast cell granules • Deep -violet • Nucleolus • Blue • Red cells • Pink • Cytoplasm of mature monocytes • Grey blue Ziehl-Neelsen staining Introduction: The Ziehl–Nieelsen stain, also called as the acid-fast stain.
It was first described by German bacteriologist Franz Ziehl and the pathologist Friedrich Neelsen. It is a special stain used to identify acid-fast organisms mainly Mycobacteria. Principle: This stain is used to identify mycobacterium tuberculosis and mycobacterium leprae (also called as acid-fast organisms). They are stained with red dye known as carbol fuschin.
Their cell wall contains mycolic acid due to which they retain dye after the application of acid. Required materials: Following materials are required; • Glass slide • Inoculating loop • Sample • Burner • Basic dye (carbol fuschin) • Heat (mordant) • Decolorizer • Counter stain (methylene blue) Procedure: Following steps are involve; • Make a smear of the specimen on the glass slide. • Fix either by heating or methanol. • Pour carbol fuschin on the smear. • Wait for 5 minutes.
• Wash with tap water. • Add 20% sulphuric acid. • Wait for one minute. • Wash off with tap water. • add methylene blue. • Wait for two minutes.
• Wash with tap water • Air dry. • Examine under microscope with oil immersion lens. Results: Acid fast bacilli stain pink. They may be straight or slightly curved rods. The background will appear blue due to methylene blue.
Interpretations: If definite bacilli are seen, report as AFB positive. S.r.no. No. of AFBs Results 1 ; 10 AFB/high power field –;+++ 2 1-10 AFB/high power field –; ++ 3 10-100 AFB/100 high power fields –; + 4 1-9 AFB/100 high power fields –; exact number Figures of staining: Giemsa stain Z.N. stain Gram stain Urinalysis: Introduction: Urinalysis is a chemical examination for identification of protein, glucose, blood cells, pH, urobilinogen, bilirubin, nitrites, ketone bodies and leukocyte esterase in urine sample. Principle: The Diagnostic Strips which are used to examine urine sample. These plastic strips contain several reagent areas separately.
Urinalysis gives information regarding the status of kidney function, liver function, carbohydrate metabolism, urinary tract infection and acid-base balance. Required materials: Following materials are required; • Urine sample • Urine stripe • Gloves Procedure: Following steps are involve; • Place urine sample at balance surface. • Wear gloves and face mask. • Take a stripe.
• Dip the stripe for one minutes into urine sample until the all reagent areas dip into the sample. • Wait for 2 minutes. • Observe the color of reagent areas. • Compare with chromatic scale. • Note the results.
Normal Values: • Color – Yellow (light/pale to dark/deep amber) • Clarity/turbidity – Clear or cloudy ? • pH – 4.5-8. • Specific gravity – 1.005-1.025. • Glucose – ?130 mg/d. • Ketones – None.
• Nitrites – Negative. • Leukocyte esterase – Negative. Oxidase test Introduction: This test is used for identification of bacteria that produce cytochrome c oxidase enzyme. If cytochrome c oxidase is present in sample, it oxidizes tetramethyl-p-phenylenediamine into indophenols (product of purple color). Principle: When the sample is applied on the oxidase paper, it turned into purple color which is positive test.
If cytochrome c oxidase is not present in sample, paper remains white which indicates to negative test. Required materials: • Moist filter paper with 1% tetramethyl-p-phenylenediamine dihydrochloride. • sample. • Pipet Procedure: Following steps are involve; • Take a filter paper.
• moiest with tetramethyl-p-phenylenediamine dihydrochloride • Socked the paper with sterile distilled water • Pick the bacterial colony with inoculating loop. • Spread on the filter paper. • Wait for 10-30 second. • Examine smeared area of paper.
Results: If cytochrome c oxidase is present, then the color will be change into purple or deep blue, otherwise it remains white in color. Catalase test Introduction: This test is used to identify catalase enzyme producing bacteria. Catalase is an enzyme which is secreted by bacteria to neutralize the bactericidal effect of H2O2. If catalase enzyme is present in sample, by applying H2O2 it produces bubbles which is the indication of positive test.
Principle: Catalase is an enzyme which is present in some types of bacteria that converts to hydrogen peroxide into oxygen and water. The bacteria that contain catalase enzyme are either aerobic or facultative anaerobes. Required materials: Following materials are required; • Glass slide. • Sample.
• Inoculating loop. • Hydrogen per oxide. Procedure: Following steps are involve; • Take a clean and dry glass slide. • Pour a small bacterial colony on the glass slide with inoculating loop. • Add a drop of H2O2 on to the slide.
• Mix well. • Wait for 5-10 seconds. • Observe the slide. Results: • If bubbling appears, the test will be positive.
• If there is no bubble or a few scattered bubbles are present, it is the indication of negative test. Coagulase test Introduction: This test is used for the differentiation between Staphylococcus aureus (positive) and Coagulase Negative Staphylococcus. Principle: The coagulase secreted by Staphylococcus Aureus reacts with factor (CRF) in plasma and makes a complex, known as thrombin. This converts fibrinogen to fibrin which causes clotting of plasma. Required materials: Following materials are required; • Sample. • Clean glass slide.
• Inoculating loop. • Normal saline. Procedure: Following steps are involve; • Take a clean and grease free slide. • Label to both sides of glass slide as “test” and “control”. • Pour a small colony on the both labelled sides of the glass slide. • Mix a drop of plasma on this suspension.
• Wait for 5-10 seconds. • Observe the slide. Results: If agglutination of cocci is occurred within 5-10 seconds, it is indication of positive test. Oxidase test Coagulase test