Medical Microbiology Techniques (ML 302) PDF

MEDICAL MICROBIOLOGY TECHNIQUES (ML 302) Unit 1: Microbiology practice and quality assurance 1 SPECIMEN COLLECTION AND TRANSPORTATION 2  INTRODUCTION Specimen collection in Microbi...

MEDICAL MICROBIOLOGY TECHNIQUES (ML 302) Unit 1: Microbiology practice and quality assurance 1 SPECIMEN COLLECTION AND TRANSPORTATION 2  INTRODUCTION Specimen collection in Microbiology is done to isolate and identify the causative agents Successful laboratory diagnosis of a microbial infection depends on many factors beginning with a well-collected sample Proper specimen selection, collection, and transport are all essential to ensure that a specimen is representative of the disease process and minimally contaminated with microorganisms present in adjacent tissues 3  SITE AND TIMING Collect the sample from the correct anatomic site, e.g. A superficial sample of a lesion is not useful in identifying the cause of a deep wound infection. The timing of sample collection is also important. E.g. when submitting a specimen for bacterial culture, samples should be collected before the administration of antibiotics, 4  Sterile technique and equipment Sufficient volume After collection, the specimen COLLECTION must be placed in an TECHNIQUES appropriately labelled leak-proof container 5  TRANSPORT OF SPECIMENS Rapidly, optimally in less than 2 hours for delays in transport, most specimens should be refrigerated All specimens for viral culture Sputum, lower respiratory specimens, stool, and urine Exceptions: blood, cerebrospinal fluid (CSF), and specimens to be examined for anaerobes, fastidious organisms such as Neisseria gonorrhoea They should be maintained at room temperature 6  SPECIMEN REJECTION CRITERIA Improper transport temperature Inappropriate specimen for test Improper transport container or requested medium Inadequate volume Prolonged transport time Specimen in fixative (for culture) Unlabelled or mislabelled specimen Duplicate sample in 24-hour Broken or cracked container period (for urine, sputum, faeces culture) Leaking specimen Dried-out specimen 7  SPECIMEN REJECTION CRITERIA When specimens are rejected, the healthcare provider is notified so that another specimen may be properly submitted If the information on the requisition is incomplete, laboratory personnel should ask a responsible person to provide the information before processing the specimen further If a specimen is mislabelled, the sample should be recollected. Relabelling of a specimen is acceptable only for difficult-to-collect specimens, such as tissue obtained during a surgical procedure or CSF 8  All specimens should be presumed to contain transmissible agents and therefore should be collected and handled using standard precautions Use of gloves, gown, mask and protective STANDARD eyewear when there is a risk of coming in PRECAUTIONS contact with the specimen In most clinical laboratories, a special area is designed for processing clinical samples for culture 9 SPECIFIC RECOMMENDATIONS FOR EACH SPECIMEN TYPE 10  BLOOD SPECIMEN COLLECTION In general, blood for culture should be obtained using an intravascular device. When performing a venipuncture, the skin must be adequately disinfected to minimize contamination with normal skin flora. Blood should be collected and incubated using the same needle into the blood culture bottles. 11  Blood specimens should be collected before administering antimicrobial agents. Optimally, the specimen should be collected just before a fever spike; however, practically, the specimen should be collected immediately after the spike. For adults, 20 to 30mL of blood should be collected per venipuncture. Less blood is required for children. 12  For adult patients, two sets of cultures should be collected per febrile episode to help distinguish probable pathogens from possible contaminants No more than four sets should be submitted in a 24-hour period. Inoculated blood culture vials should be held at room temperature until they reach the laboratory. 13 CSF – SPECIMEN COLLECTION 14  Obtained by lumbar spinal puncture Generally, at least 0.5mL of CSF (smear, culture, antigen tests ) For mycobacterial culture, at least 3mL (greater volumes increase recovery) Transported to the laboratory promptly and processed as soon as possible. If a delay in processing is unavoidable, the specimen should be held at room temperature. If greater than 1.0mL of CSF is received for a given test the fluid is centrifuged to allow the test to be performed on the concentrated sediment 15  GASTROINTESTINAL TRACT-SPECIMEN COLLECTION 16  Used to detect bacterial, viral or parasitic infections Feces, and in some cases rectal swabs (usually unsatisfactory), are submitted to the laboratory primarily to determine the etiologic agent infections diarrhea or food poisoning. Feces should be collected in a clean container with a tight lid and should not be contaminated with urine, barium, or toilet paper. Optimally be examined within 2 hours of collection. Rectal swabs should be placed in a tube transport system containing modified Stuart’s medium. Unpreserved stool specimens should be maintained at refrigerator temperature during storage and transport. 17  Collect 1 – 2 ml of faeces and apply the cap tightly Take care not to soil the rim or outside of the bottle Transmit the container quickly to laboratory If delay is unavoidable and particularly when the weather is warm collect the specimens in a container holding 6 ml buffered glycerol saline transport medium It is becoming standard practice to reject stool specimens for bacterial culture and parasite examination from patients who have been hospitalized longer than 3 days. For such patients, examination for the toxins produced by Clostridium difficile is recommended. 18 GENITAL TRACT – SPECIMEN COLLECTION 19 Genital tract specimens are sent to the laboratory for determining the cause of various clinical syndromes, including vulvovaginitis, bacterial vaginosis, etc. Many specimens will be contaminated with the normal microbiota of the genital tract or skin; therefore, the microbiologist must differentiate the normal flora from potential pathogens. Organisms such as N. gonorrhoeae, C. trachomatis, and Haemophilus ducreyi are always pathogenic, whereas organisms such as the Enterobacteriaceae, S. aureus, and group B streptococci are pathogenic only in some clinical situations. 20  The specimen commonly collected for the diagnosis of vaginitis, vaginosis or urine sepsis is high vaginal swab The swab is inserted into upper part of the vagina and rotated there before withdrawing it 21  Endocervical swab used for examination of gonococci A vaginal speculum must be used to provide a clear sight of the cervix and swab is rubbed in and around the opening of the cervix and withdrawn without contamination from vaginal wall Other swabs should be collected from any exudate discharged from the meatus of the urethra Consider rectal swab depending on sexual habits of the patient All swabs to be promptly transported to laboratory, in cases of delay or in cases of delicate microbes to be transported in Amie’s transport medium If possible two swabs to be collected and submitted for each site 22  SPECIMEN COLLECTION IN MEN Infection is mostly caused by the same organism as in women Urethritis can be caused by gonococci and or non-gonococcal infection For gonococcus infection in men Specimen is collected by milking the urethra Urethral discharge is smeared on slides and inoculated on warmed plates of heated blood agar or selective medium for isolation of Gonococci Exudates are collected for examination of chancre 23 RESPIRATORY TRACT – SPECIMEN COLLECTION 24  primarily to determine the etiologic agent of pneumonia Specimen types: LOWER sputum (expectorated or induced), tracheal RESPIRATORY aspirates, transtracheal aspirates, bronchial washes, TRACT bronchial brushings, and bronchoalveolar lavage fluids. delivered promptly to the laboratory. If delays are unavoidable, refrigerate. 25  Specimens are collected in a sterile screw cap container Its always better to have patient brush teeth and then rinse or gargle with water before collection and ideally before antibiotics are given The most common method of collection is SPUTUM expectoration from a cooperative patient with a CULTURE productive cough Early morning is the optimal time to collect sputum specimens A sputum specimen can be collected in a sputum trap from patients who have artificial airways and require suctioning 26  Precautions Avoid spilling material over the rim Tightly screw on the cap of the container Wipe off any spilled material on its outside with tissue paper Deliver the specimen quickly to laboratory 27  Nasopharyngeal aspirates, washings, and swab specimens are primarily used for the diagnosis of viral respiratory infections but may also be submitted to diagnose pertussis, diphtheria, chlamydia infections, and candidiasis, as well as identify carriers of N. meningitidis or S. UPPER aureus. RESPIRATORY Throat swab specimens are generally collected TRACT to diagnose group A streptococcal pharyngitis or to detect shedding of viruses such as enteroviruses, HSV, or CMV. Specimen should be stored at room temperature 28  An adequate view of the throat should be ensured by good lighting conditions and the use of a disposable wooden spatula or a tongue depressor to pull outwards and so depress the tongue 29  Swab should be replaced in its tube with care not to soil the rim If it cannot be transported immediately to the lab it should be placed in a refrigerator at 4°C or preferable submitted in a tube of transport medium 30  NASAL SPECIMENS A deep nasal swab generally yields the same information as throat swab Nasal swabs are taken to diagnose S. pyogenes and Diphtheria bacillus 31 TISSUES – SPECIMEN COLLECTION 32  TISSUES procured at great expense and considerable risk to the patient; therefore, for optimal evaluation, enough material should be collected to allow both histopathologic and microbiologic examination. Can be done by needle biopsy at the site of infection to collect cells and/or tissue After collection, tissues should be placed in a sterile container and transported rapidly to the laboratory to prevent drying. 33 URINE – SPECIMEN COLLECTION 34  Acceptable methods of urine collection include midstream clean catch, catheterization, and suprapubic aspiration. Foley catheter tips should not be accepted for culture. Promptly taken to the laboratory and processed within 2 hours of collection. If delays are unavoidable, refrigerated. Always give a written instruction for the patient to avoid contamination during collection 35 SKIN AND SUBCUTANEOUS LESIONS – SPECIMEN COLLECTION 36  Ideally, the infected material is aspirated with a needle and syringe. For transport, the material is expelled into sterile container that is tightly capped and promptly delivered to the laboratory. If an aspirate cannot be obtained, swab specimens of exudate collected from the deep portion of the lesion are acceptable. For bacterial and fungal cultures, swabs may be placed in tube transport system containing modified Stuart’s medium. 37  To recover anaerobes, an additional swab specimen must be collected and placed in an anaerobic transport system. For viral culture, the specimen (aspirate or swab) should be placed in viral transport medium and kept on ice. If a delay in processing is unavoidable, specimens may be stored in the refrigerator, except those for recovery of anaerobes (room temperature ) 38  WOUND CULTURE The ideal sample is pus or exudates They should be submitted in a small screw-capped bottle in firmly stoppered tube or syringe or a sealed capillary tube Superficial wound Wipe the area with sterile saline or 70% alcohol, swab along leading edge of wound Collect as much exudate as possible from the advancing margin of the lesion Avoid swabbing surround skin 39  Deep wound Wipe the area with sterile saline or 70% alcohol Aspirate material from the wall of excise tissue 40 QC/QA BACTERIOLOGY LABORATORY 41  INTRODUCTION Clinical microbiology procedures are somewhat different than those in other areas of the laboratory The subjective nature of microbiology testing requires that the technologist depend more on his experience and knowledge to make independent judgments when identifying microorganisms Obtaining an adequate specimen and utilising to quality materials to perform the testing are essential for accurately identifying microorganisms and for providing the clinician with important, timely information necessary for treating his patient 42  Quality assurance procedures involve: Maintaining a competent level of expertise among the technical staff Assuring the quality of the specimen The quality control of media, reagents and stains External quality control of laboratory by participating in proficiency surveys Maintaining laboratory equipment 43  TECHNOLOGIST TRAINING AND CONTINUING EDUCATION It is essential that all technologists have a fundamentally sound educational background in microbiology before being employed in a clinical microbiology lab. Once hired, the Lab. Management should provide adequate materials and continuing educational opportunities to ensure that competence and motivation remain high Microbiology journals and clinical case studies can be used in these programs These programs should be routinely scheduled and should involve the participation of the technical staff and laboratory pathologist They keep the technologists up- to-date with new procedures, skills sharpened, and also help keep motivation at a high level 44  It serves as a reference document that outlines the basic protocols and procedures for the analysis of the microbiological specimen laboratory Safety should be emphasized stressing the infectious, chemical, and electrical hazards of every procedures SOP should emphasize the following: PROCEDURE MANUAL 1. Sample collection and transportation to the lab Different specimen types such as swabs, fluids, tissue samples, aerobic and anaerobic culture will reach to the Lab The procedures of how to collect and transport the sample must be available to the physicians and nursing staff 45  Criteria for rejection of improper specimens List all the of the conditions that would result in a specimen being rejected by the laboratory. Specimens that are incorrectly labelled Received in an incorrect container Improperly transported Protocols for plating specimens: Procedures for the plating of specimens, both aerobically and anaerobically, The selection of media which will be used for each of specimen The incubation and preparation of CO2 and anaerobe jars should be described Outline of examination procedures for cultures: Step- by- step procedures for the macroscopic and microscopic examinations of cultures and guidelines for interpreting and reporting of results 46  Procedures for the performance of differential procedures Step - by - step describe the biochemical test and stains necessary for identifying microorganisms that have been grown from culture Information should include: The required specimen type Instructions for the macroscopic and microscopic inspection of the colony (culture characteristics) The specific media or reagents that should be used Ways to perform the test Ways to interpret the results (e.g color changes or turbidity changes) Ways to report the results The appropriate quality control procedures. 47  Antimicrobial susceptibility testing procedures Antibiotics that should be used How to measure the inhibition zone and determine the sensitive and resistant reactions The quality control organism that should be used to monitor the antibiotic inhibition, the tolerance limits Procedures for preparing media and reagents This include the quality control procedures for all media, agars, and reagents either prepared in the laboratory or commercially obtained 48  Quality assurance of laboratory equipment The maintenance procedures for equipment essential to microbiology testing (e.g refrigerators, freezers, thermometers, incubators, anaerobic chambers, autoclaves, microscopes and centrifuges) must be mentioned Procedures for handling and disposing of contaminated materials 49  QUALITY ASSURANCE OF MICROBIOLOGICAL SPECIMENS All the specimens must be collected under aseptic technique as possible as to prevent contamination with normal flora or other organisms As a general rule specimens should be received within 60 minutes of collection unless an appropriate transport medium is used Microorganisms are sensitive to environmental changes Steps should be taken preserve the integrity of the specimen once it has been removed from the body, to prevent microorganisms from dying during transportation to the laboratory 50  Before a sample is accepted for analysis, it is important that the laboratory insist that the original condition of the sample and its container be maintained There should also be adequate documentation stating the samples source, date and time of collection, analysis requirements and required storage conditions 51  QUALITY CONTROL OF MEDIA: PURCHASED MEDIA Conditions encountered during shipment can change the properties of media The purchased media should be tested for sterility and performance when received at the laboratory using test organisms that are known to produce a specific reaction on the media in question 52  QUALITY CONTROL OF MEDIA: LABORATORY PREPARED MEDIA The following recommendations are presented for laboratory preparation of media: 1. Water: Distilled or deionized water should be used pH must be checked daily, should be maintained between (5.8 and 7.0) The water should be sterile 2. Opened dehydrated products: When dehydrated media is received in the laboratory it should be marked with the day on which it was received and the day on which it was opened Store the media under the condition specified by the manufacturer and keep it tightly sealed 53 3. Sterilization: Media must be sterilized using autoclaving Not suitable for heat-sensitive components (sugars such as glucose and sucrose), these sugars should be autoclaved at 110 C° for 30 minutes After sterilization randomly remove plates or tubes of media and incubate to check and see if there has been contamination 4. pH check: After the media has been sterilized and cooled, it should be checked to see that pH is within acceptable ranges If not the entire batch should be discarded 54 5. Storage Store the prepared, sterilized media according to the manufactures recommendations The information that should be recorded on laboratory prepared media include: Expiration date, and lot number In some cases it will be necessary to label the media with its name to prevent it from being confused with other similar appearing media All records and measurements taken during the course of the preparation should be permanently recorded and kept Date of preparation pH and resistivity of rehydrating water Autoclave performance and function checks (e.g temp , pressures , and sterility checks ). 55  PERFORMANCE TESTING OF MEDIA- REAGENTS AND STAINS All lots of media (commercially & lab. Prepared), all reagents, and stains should be tested for performance upon receipt To test proper performance, the laboratory must maintain a collection of stock cultures of strains of bacteria New lots and shipments of reagents and stains should also be checked for performance with specific organisms upon receipt and at daily or weekly intervals depending on stability For media, remove from storage and incubate them at the conditions under which the test will be performed Discard the entire lot of media if there appears to be contamination 56  ANTIMICROBIAL SUSCEPTIBILITY TESTS 1. Agar diffusion method Discs used for agar diffusion susceptibility should be stored at -20°C in a desiccated container A working supply of disks can be stored at 5°C also in a desiccated container The discs should not be used after exp. Date Three organisms are recommended for performance testing E. coli, Staphylococcus aurous and Pseudomonas aeruginosa. Inoculate and incubate an agar plate with disks for each organism and measure the size of the zone of inhibition Acceptable zone size ranges are shown in Table. 57 58 2. Minimal inhibitory concentration test (MIC) MIC is defined as the lowest concentration of antimicrobial agent required to inhibit growth of the bacteria The drugs used in this antimicrobial susceptibility test should be made from pure assayed materials stored under desiccation until time of preparation It is not recommended that clinical preparations of these drugs be used but that the pure powdered drug be obtained from a pharmaceutical supply house (or similar source), carefully weighed to four decimal places, and then accurately diluted 59  Performance testing of these dilutions can be performed with one or more of the following organisms: E. coli, Enterococcus, S. aureus, and P. aeruginosa A control organism should be run with every batch of tests to monitor the potency of the antibiotics and the accuracy of the dilutions 60  Participation is proficiency surveys and testing programs: by laboratories performing biological testing is required by various local, state, federal, and accrediting agencies These surveys are used as a means of evaluating the performance of a laboratory in EXTERNAL QUALITY identifying microorganism sent in survey CONTROL – specimens and provides an evaluation of the PROFICIENCY equipment, media, reagents for differential SURVEYS tests, stains, and proficiency of the technical personnel Proficiency surveys do not evaluate the acceptability of specimen collection and transport of the specimen to the laboratory 61  Specimens are received in the laboratory in lyophilized media that needs to be reconstituted with sterile broth or water After the specimen is reconstituted, it is inoculated into the appropriate growth medium After incubation, the specimen is inoculated onto culture plates and differential tests or is Gram stained After the organism (or organisms ) have been identified, the results are recorded on the survey report form and mailed back to the coordinator of the survey Each organism found in the survey should be saved by culturing on the appropriate storage media 62  The correct or "true" identification of organisms for each specimen has been determined beforehand by referee laboratories Acceptable performance is the correct identification (that is , agreement with the referees ) of all organisms in the survey sample All unacceptable performances should be investigated by replanting the specimens, and resolution of all unacceptable results For optimal use , these specimens should be entered into the laboratory as blind samples, to be treated as patient specimens The specimens must be placed in a form that is indistinguishable from patient samples, with a fictitious name and hospital identification number Care must be taken to prevent the results from being 63 reported as patient results  SOURCES OF ERROR IN MICROBIOLOGY 1. Improper storage of media, both in the unprepared and final form 2. Using outdated media and reagents 3. Incorrectly weighing dry materials or measuring water in reconstituting media and reagents 4. Using tap water instead of deionized or distilled water 5. Using glassware or containers that are contaminated with detergents or chemicals 6. Plating the specimen on the wrong media 7. Over decolorization of Gram stain 64  8. Accepting a specimen that is incorrectly transported, e.g., dried out, or a specimen for anaerobic culture transported aerobically 9. Mixing of results and report forms 10. Incorrectly storing purchased media 11. Failing to incubate specimen in a CO2-enriched atmosphere that requires it 65  REVIEW QUESTIONS Mention TWO things that could happen if a specimen is exposed to incorrect environmental conditions during transport What is the recommended temperature for transporting CSF samples? Mention FOUR factors that should be considered when transporting microbiological specimen How does calibration of laboratory equipment contribute to quality assurance? A 28-year-old woman presents to the clinic with symptoms of frequent urination, sensation during urination, and lower abdominal discomfort. The physician suspects a urinary tract infection and requests a urine sample for diagnostic testing Describe the proper procedure for collecting a urine sample to minimise contamination for an accurate diagnosis of UTI How should the urine sample be transported to the laboratory for testing to ensure the most reliable results? 66