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Practical 1 Instructor information 2  Table of Contents Title Page Microscope 4 Staining Techniques for Microbial 11 Examination Culture media 19 Sample Coll...

Practical 1 Instructor information 2  Table of Contents Title Page Microscope 4 Staining Techniques for Microbial 11 Examination Culture media 19 Sample Collection for Bacterial Isolation 32 Serology 43 3 Microscope 4  MICROSCOPE Bacteria are very small in size, measured in terms of microns (= 1/1000 mm). Hence they cannot be seen by the naked eye. Microscopy use of microscope as a method of examination. Key characteristics of a reliable microscope are: Magnification means the ability to enlarge objects, Resolving power means the ability to show details. Types of Microscopes: 1. Light microscope: Bright-field (Ordinary light) microscope. Dark ground "Dark field" microscope. Phase contrast microscope. Ultra-violet or "Fluorescence microscope". 2. Electron microscope. I. Ordinary Light Microscope Structure: 1. Optical System: - Outer tube: bears 3 interchanged objective lenses: - Low power, magnification power is 10x. - High power, magnification power is 40x. - Oil immersion lens, magnification power is 100x. - Inner tube - Body: 2. Stage: - The stage is fenestrated by a central aperture. 3. Sub-stage group: 5  - Condenser: for collection of light rays to fall on the object. - Iris diaphragm: for control of the amount of rays passing. - Light source: (mirror/lamp). Photo (1) Ordinary light microscope Magnification of the microscope: Total magnification = Objective Magnification x Eye piece Magnification. Microscope Resolution: Ability of lenses to distinguish two points, the shorter wavelengths, the better the resolution. 6  Cedar Oil: - Placing this oil (with the same refractive index as glass) between the objective lens and the slide decreases the number of refractive surfaces. - The oil enhances resolution by preventing light rays from dispersing after passing through the specimen. Precautions for the use of the microscope: 1. Light is turned on. 2. Iris diaphragm is widely opened. 3. Condenser is high up. 4. Oil immersion lens is used. 5. Drop of cedar-wood oil is used. II. Dark Field Microscope Used to see some refractile organisms as Spirochetes, that are delicate, so cannot be seen in presence of excess light. Special condenser oblique light only. Photo (2) Dark field microscope 7  III. Phase Contrast Microscope This type is used to examine living organisms or its detail structures e.g. flagella, capsule. IV.II. Fluorescence Dark Field Microscope Microscope The bacterial cell stained by fluorescent dye as acridine orange, we use the ultra- violet rays instead of ordinary light. Photo (3) Bacterial strain detected by fluorescence Microscope V. Electron Microscope An electron beam with very short wavelength is used instead of light. Resolution power is very minute about 1 nm, so can examine very small objects as viruses. Disadvantages of electron microscope: - Needs special experience. - Expensive. - Needs special sections (Ultra-thin section). 8  Photo (4) Electron Microscope Questions Choose the best matched answer: 1. The magnification power of the ordinary light microscope when using the OIL is: a. 10x b. 100x c. 40x d. 1000x e. 400x 2. The microscope used for examination of refractile organisms is called: a. Ordinary light microscope. b. Dark ground microscope. c. Phase contrast microscope. d. Electron microscope e. Immunoflurescent microscope 9 3. The microscope used for examination of viruses is called: a. Ordinary light microscope. b. Phase contrast microscope. c. Dark field microscope. d. Electron microscope. e. Light microscope. 4. UVR is used in: a. Light microscope. b. Dark field microscope. c. Phase contrast microscope. d. Immunofluorescence microscope. e. Electron microscope. 5. Bacterial cells examined by fluorescence microscope can be stained by: a. Giemsa stain. b. Silver fontana stain. c. Acridine orange. d. Gram stain. e. Methylene blue stain. Answer key: 1) d 2) b 3) d 4) d 5) c References: Cheesbrough M. (2000): Microbiological tests. Cited by Cheesbrough, M., (ed.) District Laboratory Practice in Tropical Countries, Part 2, Microscopical techniques used in microbiology, Cambridge University Press, UK Useful websites: https://youtu.be/mJ9_VhxJMi8 10  Staining Techniques for Microbial Examination 11  STAINING METHODS A staining technique is often applied to the bacterial cells to color them so that their shape, size and arrangement can be easily determined under the microscope. Main lines: 1. Preparation of dried and fixed film. 2. Staining of the film by a suitable stain. Smear preparation: Preparation and Fixation of Bacteria for Staining. Objective: To kill the microorganism & fix them to the slide to prevent them from being washed out during the process of staining. Preparation of dried and fixed film: 1. Sterilization of the bacteriological loop. 2. Pick a pure colony in a drop of water or bacterial culture drop. 3. Spread on a slide. 4. Drying of the smear. 5. Fixation of the smear. 6. Staining. Photo (1) Preparation of dry fixed film 12 Simple Stain Principle: - The use of one chemical dye to stain the organisms (all the organisms are stained with the same color of the dye). Uses: - Identification of the shape and arrangement of the organism. The most commonly used simple stains are: - Methylene blue. - Diluted carbolfuchsin. Procedure of staining with simple stain: - Prepare dried and fixed film. - Cover the preparation with methylene blue (or diluted carbolfuchsin) for 1-2 minute. - Remove excess stain and wash with water gently until the color is faint. 13  - Dry the preparation either in air or on the flame. - Examine with oil immersion lens. Results: - All organisms appear either blue with methylene blue, or red with carbolfuchsin. Photo (2) Simple stain of Staphylococci by methylene blue Photo (3) Different shapes and arrangement of the bacteria Differential Stain (Gram stain) Principle: - It differentiates between Gram-positive bacteria (that resist decolourization and retain the color of the basic dye which is methyl violet) and Gram-negative bacteria (that allow the crystal violet-iodine complex to be washed out easily using the decolorizer and then be stained by the color of the counterstain which is the diluted carbolfuchsin). 14  The different reaction to the Gram stain is due to: Cell wall in gram positive & negative organisms Gram-positive bacteria  Have a thick peptidoglycan layer surrounds the cell.  The stain gets trapped into this layer and the bacteria turned purple.  Retain the color of the primary stain( crystal violet )after decolorization with alcohol Gram-negative bacteria  Have a thin peptidoglycan layer that does not retain crystal violet stain.  Instead ,it has a thick lipid layer which dissolved easily upon decoulorization with Alcohol.  Therefore ,cells will be counterstained with diluted carbol fuchsin and turned red. Requirements: 1. Methyl (crystal) violet 1%, 1ry stain. 2. Iodine solution, mordant. 3. Alcohol 95%, decolorizer, 4. Diluted carbol fuchsin 0.1%/ Safranin (counter basic stain). Procedure of staining: - Prepare dry and fixed film. - Cover the preparation with methyl violet for one minute. - Wash with water. - Add iodine solution for 1 minute followed by rapid washing with water. - Decolourization. "Most important step". 15  o Add alcohol and leave it with tilting the slide for 15 seconds. o Wash with water. o Repeat the above steps until the colour of alcohol becomes faint violet. o Avoid over decolourization because Gram positive may appear negative. - Add diluted carbolfuchsin for one minute. - Wash with water. - Dry and examine the film microscopically. Results:  Gram positive organisms appear dark purple.  Gram negative organisms appear pink. Photo (4) Staphylococci in culture by Gram Photo (5) Gram positive bacilli in culture by stain Gram stain 16 Photo (6) Gram negative bacilli in culture by Gram stain Questions Choose the correct answer: 1. Simple stain gives an idea about: a. Shape and arrangement of bacteria. b. Gram reaction of bacteria. c. Presence of bacterial capsule. d. Motility of bacteria. e. Genus of bacteria. 2. The different reaction to Gram stain is due to: a. Thick peptidoglycan layer in Gram negative bacteria. b. Thin layer peptidoglycan in Gram positive bacteria c. Protoplasm of Gram negative bacteria is strong acidic. d. Permeability in Gram positive bacteria is more than in Gram negative bacteria. e. Permeability in Gram positive bacteria is less than in Gram negative bacteria. 3. The counterstain in Gram stain is: a. Methyl violet 1%. 17  b. Iodine solution. c. Alcohol 95%. d. Diluted carbolfuchsin 0.1%. e. Methylene blue. 4. Decolourization in Gram stain is done by: a. Sulphuric acid 20% b. Sulphuric acid 95%. c. Alcohol 20 %. d. Alcohol 95 %. e. Iodine solution. Answer key: 1) a 2) e 3) d 4) d References: Cheesbrough M. (2000): Microbiological tests. Cited by Cheesbrough, M., (ed.) District Laboratory Practice in Tropical Countries, Part 2, Microscopical techniques used in microbiology, Cambridge University Press, UK Useful websites: https://www.youtube.com/watch?v=JvN6t8- assk 18 19 Culture & medium: – Culture is a term given to micro-organisms that are cultivated in the lab. – Medium is a term given to the combination of ingredients that will support the cultivation of these micro-organisms by providing all the essential nutrients. – Culture medium provides the nutrients required by the bacteria for growth & multiplication MEDIA b. Culture media a. Transport media Culture media provide nutrients required by the bacteria for growth and multiplication. 20 Types of culture media: 1. ORDINARY (SIMPLE) MEDIA 2. ENRICHED MEDIA 3. SELECTIVE MEDIA 4. INDICATOR MEDIA 5. ENRICHMENT MEDIA Types: 1. ORDINARY (SIMPLE) MEDIA Media Uses Fluid Nutrient - Base for nutrient agar. Media - Culture of non-fastidious broth bacteria Solid Nutrient - Plates are used for primary Media culture of many microorganisms. Agar - Slopes are used for subculture and preservation of bacteria. - Deep agar is used for culture of anaerobic bacteria. - The medium is the base of different types of media. 21  Photo (1) Different forms of ordinary media (Uninoculated) Identification of bacterial growth on fluid media: - General turbidity if the bacteria are facultative anaerobes. - Surface pellicle if the bacteria are aerobic. - Deposits in the bottom of the tube in case of anaerobes. - Grow below the surface of the fluid in case of microaerophilics. Photo (2) Growth pattern of bacteria on fluid media. 22  2. ENRICHED MEDIA These media contain substances of highly nutritive value for fastidious bacteria. Media Uses Blood agar For the fastidious bacteria. An indicator medium i.e.help in the identification of bacteria by their haemolytic action on the red cells. Chocolate agar Culture of Neisseria and Haemophilus groups. Loffler’s serum Culture of diphtheria bacilli. Dorset’s egg medium Culture of Tubercle bacilli from non -contaminated pathological sample e.g.C.S.F. 23  Photo (3) Blood agar plate (uninoculated) Photo (4) Chocolate agar plate (uninoculated) Photo (5) Loffler serum (uninoculated) Photo (6) Dorset egg media (uninoculated) Types of haemolysis on blood agar: a) - Haemolysis (partial haemolysis): It occurs due to partial destruction of RBCs with release of biliverdin (green) e.g. Streptococcus viridans and Streptococcus Pneumoniae. b) - Haemolysis (complete): Colonies are surrounded by zones of clear haemolysis due to complete destruction of RBCs by toxins released from bacteria e.g. S. aureus & Streptococcus pyogenes. c) - Haemolysis (no haemolysis): e.g. Strept.faecalis 24  Photo (7) Different types of haemolysis on blood agar 3. SELECTIVE MEDIA These media contain some dyes or chemicals or antibiotics that inhibit the growth of certain organisms and allow the growth of others. Media Selective component Uses Lowenstein Jensen Cultivation of Tubercle bacilli medium( L.J:) Malachite green from contaminated sample e.g.sputum inhibits the growth of bacteria other than T.B. Thayer - 3 antibiotics Culture of N. Martin gonorrhoeae. medium Vancomycin ,Colistin. and Nystatin inhibit other saprophytes. 25  Photo (8) LJ medium 4. INDICATOR MEDIA These media usually contain an indicator which changes its colour as a result of the metabolic activities of particular organisms, useful for the cultivation of enteric bacteria. e.g.Enterobacteriaceae. Media Indicator Uses MacConkey’s Cultivation of enteric bacteria, Neutral red medium: Differentiate between two major groups: changes pink in the presence of LF :rose pink colonies. acid which is produced as a NLF :pale colonies result of lactose fermentation Bromothymol blue Cystein lysin Culture of urine sample: electrolyte deficient medium LF :Yellow colonies. )CLED( NLF :Pale colonies 26 Photo (9) MacConkey's agar Photo (10) LF on MacConkey's agar Photo (11) NLF on MacConkey's agar (uninoculated). Photo (12) CLED agar Photo (13) LF on CLED agar Photo (14) NLF on CLED (uninoculated). agar 5. ENRICHMENT MEDIA - These are fluid containing some substances, which stimulate the growth of some organisms on the expense of the unwanted organisms. - Example: Selenite broth: used for the isolation of the Shigella and Salmonella from faeces. 27  Photo (15): Selenite broth Maintain the viability of a pathogen during transit from the patient to the laboratory. STUART’S TRANSPORT MEDIUM: - Maintain the viability of Gonococci on swabs during their transmission to the laboratory. Photo (16) Stuart transport media 28 Multiple Choice Questions: 1. Media that inhibit the growth of certain bacteria while allowing other types of bacteria to grow is: a. Ordinary media. b. Enriched media. c. Selective media. d. Indicator media. e. Enrichment media. 2. Media that promote the growth of bacteria that might be present in a sample in low numbers is: a. Ordinary media. b. Enriched media. c. Selective media. d. Indicator media. e. Enrichment media. 3. Which one of the following is an example of ordinary media? a. Nutrient agar. b. Chocolate agar. c. Loffler’s serum. d. MacConkey's medium. e. CLED medium. 29 4. Which one of the following media is an enriched media for Neisseria species? a. Chocolate agar. b. Loffler’s serum. c. MacConkey's medium. d. CLED medium. e. Thayer- Martin medium. 5. Which is the selective substance in Lowenstein Jensen medium? a. Potassium tellurite. b. Malachite green. c. Bile salt. d. Phenol red. e. Neutral red. 6. Which is the selective substance in CLED medium? a. Malachite green. b. Bromothymol blue. c. Bile salt. d. Phenol red. e. Neutral red. 7. Which one of these is an example of transport media? a. CLED medium. b. MacConkey's medium. c. Stuart medium. d. Dorset egg. e. Litmus milk. 30 8. On MacConkey's medium E. coli forms: a. Colourless colonies. b. Greenish pigmentation. c. Yellow coloured colonies. d. Pink coloured colonies. e. Medusa head appearance. Answer key: 1) c 2) e 3) a 4) a 5) b 6) b 7) c 8) d References: Cheesbrough M. (2000): Microbiological tests. Cited by Cheesbrough, M., (ed.) District Laboratory Practice in Tropical Countries, Part 2, Microscopical techniques used in microbiology, Cambridge University Press, UK Useful websites: https//:www.youtube.com/watch?v=Yl1EZi0qX1Q 31  Sample Collection for Bacterial Isolation 32  Collection of Samples for Bacterial Isolation General rules should be applied to all specimens: 1. The samples must be taken before the start of antimicrobial therapy. 2. Hands should be washed before and after the collection. 3. The specimen must be collected aseptically in an appropriate, sterile container. 4. The Sample should be representative of infectious process 5. The quantity of specimen must be adequate. 6. The container must be dated, appropriately labeled. 7. The specimen must be transported rapidly to the laboratory and transport media may be helpful. Selected cases for sample collection: 1. Respiratory Tract infection a. Throat swab: URTI b. Nasopharynx: nasopharyngeal washing or nasopharyngeal swab in meningitis and viral infection. c. Middle ear: - Otitis media with discharging ear, the sample is taken by ordinary swab from the discharge deeply as you can. d. Lower respiratory: - Sputum is collected in sterile container; the best sample is the early morning sputum. The patient should expectorate from deep down in the lung. 33  - Trans-tracheal aspiration, lung biopsy, or bronchoalveolar lavage (this is done during examination or operation). Photo (1) Sterile disposable Photo (2) Throat swab Photo (3) Nasopharyngeal swab cotton swab 2. Acute Intestinal Infection a. Faeces: - Faeces should be passed into clean containers, and should be sent to the laboratory as soon as possible. b. Vomitus: may be taken in cases of poisoning. Photo (4) Sterile container Photo (5) Rectal swab for stool samples 34  3. Urinary Tract Infection - Urine specimens are usually collected in sterile containers. a. Mid-stream specimen: - Cleaning of the external genitalia with soap and water. - Discard first part of urine, then collect mid-stream part in sterile container. b. Adhesive bags: - Used for collection of urine specimen especially in children. c. Supra-pubic aspiration: - In infants or in case of urine retention or pregnancy. d. Catheterization of urethra or ureter: - For collection of urine if the patient is already catheterized. Photo (7) Supra pubic aspiration Photo (8) Adhesive bag Photo (6) Sterile disposable container Photo (9) Urethral catheter Photo (10) Urethral catheter (Nelaton) (Foley) 35  4. Meningitis CSF taken by lumbar puncture. – Under strict aseptic precautions to prevent introduction of infection. – The patient sits with extended vertebra by flexion forward as possible and the lumbar area is sterilized by Tr. iodine then with absolute alcohol, then with special needle puncture between 4th and 5th lumbar vertebrae. – The sample is collected in screw-capped bottles and sent to the laboratory at once. Photo (11) CSF taken by lumbar puncture Photo (12) Screw-capped bottles 5. Wounds, abscesses, fluids, tissues a. Ordinary swab: Which must be soaked well in pus or exudates deep from infected wounds. b. Aspiration by syringe: To obtain a specimen pus itself and transfer to a sterile tube. c. Pieces of tissues: Removed at operation or curettage from infected tissues, then homogenized in a tissue grinder with a little broth. 36  Photo (13) Swab deep from wounds Photo (14) Pus aspiration 6. Genital Tract Infection a. In females: - Cervicitis: Cervical swab. - Vaginitis: vaginal swab, discharge. b. In male: - Acute: urethral discharge. - Chronic: morning drop, prostatic massage. 7. Septicemia The sample collected is blood. Blood culture technique: Procedure: 1- Use only sterile equipment and strict a septic techniques. 2- Apply a tourniquet and locate a fixed vein by touch 3- Prepare the skin by 2% Tr.iodine and 70% alcohol. 4- Perform venipuncture and withdraw 10 ml blood. 5- Add blood to aerobic and anaerobic culture bottles. 6- Put in an incubator rapidly. 37  Steps: 5-10 ml of blood is added to 50 ml broth in blood culture bottles ,which are incubated at 37° C. Subcultures are made every day by withdrawing small amount (loopful) of blood broth mixture and plating it out on blood agar If results of subculture are negative for 7 successive days ,discard the blood culture. The big volume of broth has the following advantages: -Good nutrition for multiplication of the organism ,which is present in small numbers. -Dilutes out any antibiotic or antibacterial substances in the serum. Photo (15) Blood culture bottles 38  Questions Multiple Choice Questions: 1. What is the proper sample to diagnose a meningococcal carrier? a. Bronchoalveolar lavage. b. Blood sample. c. Nasopharyngeal swab. d. CSF. e. Urine sample. 2. What is the proper sample to diagnose septicemia? a. Bronchoalveolar lavage. b. Blood sample. c. Nasopharyngeal swab. d. CSF. e. Urine sample. 3. What is the proper sample to diagnose acute cervicitis in female? a. Urethral discharge. b. Vaginal swab, discharge. c. Blood sample. d. Cervical swab. e. Urine sample. 4. What is the proper sample to diagnose chronic gonorrhea in male? a. Urethral discharge. b. Morning drop, prostatic massage. c. Blood sample. d. CSF. 39  e. Urine sample. 5. The blood culture bottle should be discarded if the subcultures are repeatedly negative for: a. 2 successive days. b. 5 successive days. c. 7 successive days. d. 10 successive days. e. 14 successive days. Answer key: 1) c 2) b 3) d 4) b 5) c METHODS OF ISOLATION OF BACTERIA - Almost every procedure used in diagnostic microbiology is based on examining pure cultures, i.e. an isolated growth of a single bacterium. - This can be achieved by the plating out technique, which aims at spreading out bacteria on the surface of solid media. Each bacterium will then divide repeatedly to give rise to a separate colony. Photo (16) Plating out technique 40  ANTIBIOTIC SENSITIVITY TESTING After a pathogen is cultured, its sensitivity to specific antibiotics serves as a guide in choosing antimicrobial therapy. Disk-diffusion method - Kirby-Bauer disk-diffusion method. - Disks with exact amounts of different antimicrobial agents are placed on culture dishes inoculated with the microorganism to be tested. - The organism's growth (resistance to the drug) or lack of growth (sensitivity to the drug) is then monitored. - The size of the zone of growth inhibition is influenced by the concentration and rate of diffusion of the antibiotic on the disk. Photo (17) Antibiotic sensitivity test 41 References: Cheesbrough M. (2000): Microbiological tests. Cited by Cheesbrough, M., (ed.) District Laboratory Practice in Tropical Countries, Part 2, Microscopical techniques used in microbiology, Cambridge University Press, UK Useful websites: https://youtu.be/V-oD1gi9deY 42 SEROLOGICAL TESTS Part I 43  SEROLOGICAL TESTS Definition: It is the study of antigen-antibody reactions in vitro. Characters of the reaction: - Specific i.e. the antibody (Ab) reacts with the antigen (Ag) that induces its production. - Optimum temperature 37ºc up to 56C. - When the antigen is: o Particulate: the reaction is called agglutination. o Soluble: the reaction is called precipitation. o Toxin: toxin-antitoxin reaction develops. o Virus: virus neutralization reaction develops. o When a third substance complement may enter the reaction, it is called complement fixation reaction. - Optimal amounts of antigen and antibody are required, because increase any of them inhibit the reaction to be visible. 44  Zone phenomena Observable antigen-antibody reactions: 1. Agglutination Definition:  Antigen antibody reaction in which antigen present in particulate form, the result of the reaction is clumping of these particulate antigens.  The antigen is called agglutinogen and the antibody is called agglutinin. Types of agglutination: A. Direct agglutination: Direct reaction between antigen and antibody, occurs either on slide or in tube. 45  Slide agglutination: Tube agglutination: Rapid, simple and It is an accurate and qualitative test, used in: quantitative test, used - Identification of in: unknown organism. - Diagnosis of infectious - Blood grouping. diseases. Titre: - It is the highest dilution of the serum that gives positive reaction. - Rising titre is diagnostic. Slide agglutination Tube agglutination 46 B. Haemagglutination test:  Some viruses e.g. Influenza virus can agglutinate sheep, horse and chicken RBCs.  Inhibition of this process by virus antibody is called haemagglutination inhibition test. Positive and negative haemagglutination test 47  c. Antiglobulin agglutination (Coombs test): Detect antibodies against Rh factor Direct Coombs test: Indirect Coombs test: - Detect the Rh antibody in - detect the Rh antibody the infant for diagnosis of in the mother serum haemolytic diseases. 48  d. Passive agglutination (Latex fixation test): - Soluble antigen is fixed on inert particles as latex which act as a particulate antigen. E.g. Rheumatoid factor, CRP and ASO test. Latex fixation test 49  References Ivan Roitt, Jonathan Brostoff and Davis Male-Immunology, 3rd Ed, 1993 Jenis Kuby, Barbara A. Osborne: Textbook of Immunology, (3rd Ed.)1992. Basic Immunology by Abdul K. Abbas and Andrew H. Lichtman ROITT’S ESSENTIAL IMMUNOLOGY, Ivan M Roitt, and Peter J. Delves 3rd,8th,10th Edition. A Textbook of Microbiology by P Chakraborty Textbook of Microbiology by Ananthanarayan and Paniker Review of Medical Microbiology and Immunology by Warren Levinson Diagnostic Microbiology by Connie R. Mahon Baily and Scott diagnostic microbiology 13th edition (2014): Serologic Diagnosis of Infectious Diseases (chapter 10, p 142). https://universe84a.com/agglutination/ 50 Serology part 2 51  2. Precipitation Definition:  This is an Antigen-antibody reaction in which the antigen is in soluble form.  In this case antigen is called precipitinogen and antibody is called precipitin. Types of precipitation tests: A. Ring test: - A solution of antigen is layered on the surface of the antibody in a capillary tube - A narrow ring of precipitate occurs near the junction of two fluids. - This test can be used for: Grouping of bacteria. Determining unknown proteins. 52  Ring test B. Agar diffusion: Antigen and antibody are allowed to diffuse against each other in agar gel 1.Single diffusion  The Ag is placed in a well and diffuses in the agar gel containing suitable ab.  A ring of precipitate forms where the reactants meet.  This test is used for quantitative determination of: Immunoglobulins, Complement components. 53  Single immunodiffusion test 2. Double diffusion:  Antigen and antibody are allowed to diffuse towards each other in an agar medium, e.g. from separate wells in an agar plate.  When antigen and antibody meet in optimal proportions they produce a thin line of precipitate.  This test is used for: Diagnosing various bacterial, viral, fungal and autoimmune diseases. Recognizing toxin production by C. diphtheriae (Elek’s test). 54  Double immunodiffusion test 3. Complement Fixation Complement is a protein fixed during the interaction of antigen and antibody. 55  Procedure: a. Test system: b. Indicator system: - Used for detection of antibodies in - Consists of the serum by adding the antigen sheep RBCs and and complement. its specific - If antibody is present, antigen antibodies antibody complex is formed and will fix complement. Results: - No haemolysis :means positive test ,antibody present in the patient serum ,the complement is fixed ,so no complement is available to cause lysis of RBCs. - Haemolysis: means negative test ,no antibody present in the patient serum ,the complement is free and haemolysis occurs. Clinical application of Complement Fixation Test: Wassermann test for diagnosis of syphilis. 56 57  4. Immunofluorescence It is an antigen-antibody reaction where the antibodies are labeled with a fluorescent dye and the antigen- antibody complex is visualized using ultra-violet (fluorescent) microscope. Types: a. Direct Immunofluorescence: b. Indirect immunofluorescence: - Used to detect antigen (viral, - This is used to detect antibodies in parasitic, tumor antigens). patient serum eg anti-nuclear antibodies (ANA) found in the serum of patients with SLE. 58  Immunofluorescence by Immunoflurescent microscope 5. Enzyme linked immunosorbent assay (ELISA)  An enzyme is linked to an antibody  It is used either to demonstrate unknown antigens or unknown antibody. Types: (Sandwich technique) (Designed to detect the a. Direct ELISA: antigens)  Antibody specific for the antigen is coated on a plastic surface  The patient serum is added to the plate.  Then a secondary antibody is added.If the antigen is present ,a“ sandwich ”of antibody ,antigen ,and secondary antibody will form. 59   The secondary antibody is linked to an enzyme.  When the substrate is added, the enzyme converts the substrate to a colored compound. b. Indirect ELISA: (Designed to detect antibodies) 60  References Baily and Scott diagnostic microbiology 13th edition (2014): Serologic Diagnosis of Infectious Diseases (chapter 10, p 142) https://www.proprofs.com/quiz- school/quizshow.php?title=basic-immunology-and- serology&q=8 Choice: 1. Agglutination reaction is more sensitive than precipitation for the detection of: a. antigens b. antibodies c. complement d. antigen-antibody complexes e. Antibody-complement complexes 2. Precipitation reaction is relatively less sensitive for the detection of: a. antigens b. antigen-antibody complexes c. antibodies d. Complement e. Antibody-complement complexes 61 3. In agglutination reactions, the antigen is a …. And in precipitation reactions, the antigen is a ….? a. Whole cell/soluble molecule. b. Soluble molecule/whole cell. c. Bacterium/ virus. d. Protein/ carbohydrate. e. Lipid/ carbohydrate. 1 2 3 a c a 62 Quiz: 1. Write the difference between the following: a. active and passive agglutination. b. agglutination and precipitation. C. direct and indirect coomb's test. 2. Why would a doctor order a direct coomb's test when a baby is born with jaundice? 3. The complement fixation test looks for the presence of specific antigens in the patients' blood a. true b. false 63