Laboratory Identification of Microorganisms PDF

Laboratory identification of microorganisms (1) preparation of the patient (2) collection of the specimen (3) processing the specimen (4) storing and/or transporting the specimen 1. Preparing the Patient appr...

Laboratory identification of microorganisms (1) preparation of the patient (2) collection of the specimen (3) processing the specimen (4) storing and/or transporting the specimen 1. Preparing the Patient appropriate collection instructions information on fasting, diet, and medication restrictions Exercise increase in blood glucose, lactic acid, serum proteins Emotional or Physical Stress Time of Day of Collection. growth hormone high in the morning Serum iron levels may change as much as 30% to 50%, from morning until evening 1  Laboratory identification of microorganisms 2. Collection of samples Bacterial culture collected prior to the start of antibiotic treatment. Actual pus or tissue samples are always preferable to swabs. Use aseptic technique during specimen collection to avoid contamination. Specimens must be collected into sterile containers. Ensure the correct specimen container is used for the sample type. Specimens must be clearly labelled. Ensure specimen container lid is secure and place in a sealable bag. Do not put samples from different patients in the same specimen bag. Samples should be transported to the laboratory as soon as possible. Most specimens must be processed within 24 hours of being taken (maximum 48 hours). 2  Laboratory identification of microorganisms Safety considerations Universal guidelines ……………. Treat all samples as dangerous Appropriate protection………… gloves, coats gowns, eyewear, face mask. Avoid surface contamination of the tubes or surroundings. Minimize direct handling of specimens in transportation to the lab. Label high risk samples: Sputum with suspected Tuberculosis Fecal samples Label High risk Specimens suspected with Cholera, Typhoid Serum when suspected with HIV/ HBV/HCV 3  Laboratory identification of microorganisms Rejected samples: Request form Leaking/broken container Name, Age, Sex, Insufficient amount Address Improper labelling IPD/ OPD More time lag between collection and transport Time and Date Improper transport media Urgent / Routine Improper transport temperature Type of specimen Hemolysed sample Investigation needed History of patient Samples(collected Provisional diagnosis Doctor’s name and sign 4  Laboratory identification of microorganisms Samples to be collected Blood Cerebrospinal fluid (Csf) Urine Mycology samples Throat Wound swabs Stool Water sample Rectal swab Work area sample Genital swab Room sample Sputum Wall samples Serum Ceiling samples Plasma 5  Laboratory identification of microorganisms 1. Phenotypic- morphology (micro and macroscopic) 1. Culture 2. Identification 3. Staining 4. Biochemical testing 5. Antimicrobial susceptibility 2. Immunological- serological analysis 3. Genotypic- genetic techniques (molecular techniques) 6 7  Laboratory identification of microorganisms Uses of bacterial culture media: Bioassay of naturally occurring Isolation of bacteria from various samples. substances as vitamins and Isolation of bacteria from mixed population. antibiotics. Differentiation between bacteria according to the Assay for antimicrobial sensitivity. macroscopic appearance of the colony and biochemical reactions. Characterization and identification of bacteria. Enumeration of the bacteria in different samples: water, sewage, food, dairy products.....etc. 8  Laboratory identification of microorganisms Types of culture media B. According to origin: A. According to consistency: 1. Natural medium 1. Liquid medium (broth) 2. Synthetic medium 2. Semisolid medium: broth containing 3. Semisynthetic medium (natural and 0.4 % agar. synthetic) 3. Solid medium: broth containing 1.5 C. According to constituents: % agar. 1. Ordinary medium 4. Firm medium: broth containing 4-6 2. Enriched medium % agar. 3. Selective medium 4. Differential medium 5. Specific medium 6. Indicator medium 9  Bacterial identification Laboratory identification of microorganisms 1. Gross colony characteristics: a. Size of colony; diameter in nm. 2. Hemolysis on blood agar medium: b. Form; punctiform, circular, irregular, 1. Alpha hemolysis: colonies are filamentous. surrounded by greenish discoloration. c. Elevation; flat, raised, convex, umbonate, 2. Beta hemolysis: colonies are surrounded umbilicate. by a zone of complete clearing of blood d. Margin (edge); entire, undulant, lobate, due to lysis or RBCs. curled. 3. Gamma hemolysis: no change in the e. Color; white, yellow, black, orange. medium around the colony. f. Surface; glistening, dull. g. Consistency; viscid, membrane, brittle. 10  Laboratory identification of microorganisms 3. Pigment production in nutrient agar 4. Odor: medium: Grape juice: Pseudomonas species. Endopigment production: bacterial Burned chocolate: Proteus species. colonies are pigmented, while the Fecal, putrid: Clostridium species. medium is normal. Exopigment production: the bacterial 5. Morphological characteristics of bacteria colonies and the medium are colored. Are studied through bacterial staining to detect morphology, shape, size, arrangements, and structure. 11  Staining of bacterial film Laboratory identification of microorganisms 2. Differential stain: a. Basic dyes: if the color is in the positive ion. more than one stain and used for differentiation b. Acidic dyes: if the color is in the of bacteria. negatively charged ion. Types of stains: a. Gram’s stain. 1. Simple stain: b. Acid-fast stain. one type of stain and used for detection of 3. Special stain: bacterial morphology. used for staining of special part of the bacteria. a. Crystal violet. a. Spore stain b. Methylene blue. b. Flagella stain c. Loffler’s methylene blue. c. Capsule stain d. Polychrome methylene blue. d. Cell wall stain 12 Laboratory identification of microorganisms Sample processing for microbial identification Antimicrobial Susceptibility Testing 13  Laboratory identification of microorganisms Sample processing for microbial identification Failure to confirm a pathogen in a specimen may indicate that: 1. The pathogen was absent or very very few in that specimen. 2. The sample was taken at a stage of the disease when the pathogen was limited in presence. 3. Viability was lost between the times of collection and arrival in the laboratory. 4. The pathogen cannot be detected by the method of testing used. 14