DEPARTMENT OF MICROBIOLOGY GENERAL INSRUCTIONS FOR COLLECTION OF DIFFERENT MICROBIOLOGY SAMPLES Good quality laboratory services means good quality medicine and better patient care. The Quality of a clinical microbiology report is directly related to the quality of the specimen submitted. Proper specimen and its prompt submission to the laboratory are essential. Note: All samples for culture should be collected in sterile containers only. The containers must be adequately labeled and sent with microbiology requisition form duly signed and filled completely with information in respect of: o Name, Age, Sex o Ward/OPD No. o Dr. In charge o Date of admission o Date & time of sample collection o Duration of Hospital stay o Antibiotic history o Last change of antibiotic o Relevant clinical history o Radiological findings o Provisional diagnosis. o Associated comorbities. Incomplete forms are liable to be rejected & reports of incomplete forms will not be released. Overflowing samples (Urine) and samples with insufficient quantity shall not be received. Microbiology department is open all 365 days a year.
Routine samples collection timings: Monday to Friday-9:00AM to 11:30AM Saturday 9:00AM to 10:30AM 1) ROUTINE SAMPLES Common collection centre: Samples for Virology, Immunology & Serology VDRL, TPHA, Widal, Rheumatoid factor (RF), ASO, CRP, AntiNuclear Antibodies (ANA), Hepatitis Viral markers (HBs Ag, Antibodies for HAV, HEV & HCV) Blood samples for Malaria antigen, Filarial antigen & other rapid blood tests. Blood culture Samples for OPD patients. Department of Microbiology All microbiology investigation requests from indoor cases. Samples for ELISA, ELISA for TORCH 2 nd & 4 th Monday Samples for HIV testing and CD4 /CD8 counts (ICTC Centre). 2) EMERGENCY SAMPLES (are received in the department only) Apart from routine sample timings All working days 9:00AM to 8:00PM Sundays & Holidays 9:00AM to 3:00PM Emergency samples: Samples collected intra- operatively, Blood culture, Throat swab for Diphtheria, Stool for V. cholera, CSF culture, gas gangrene and samples pertaining to any acute infection outbreaks. 3) SPUTUM CULTURE Early morning coughed up sputum (Not Saliva) sample. Collect in a sterile wide mouthed container with screw tight cover. For Gram stain request of sputum sample: it is required to be clearly spelt out as to the type of organism/disease that is being strongly suspected. Without this information Gram stain of sputum sample may not have any clinical significance 4) THROAT SWAB Collect the sample with sterile swab by swabbing tonsillar area, posterior pharyngeal wall or inflamed area of the throat without touching the oral area. Try to include the pus or membrane if any. Send two swabs, one for culture and other for staining. 5) BLOOD CULTURE
Give details of duration of hospital stay, day of onset of fever, duration of fever, antibiotic therapy, whether sample collected directly or from canula, earlier report of organism isolated & its sensitivity. Collection procedure: clean the site from inward to outwards in a circular pattern, first with 70% alcohol, let it dry, then clean with povidone iodine & collect venous blood. Send in BHI broth provided by department of Microbiology. Amount Adult - 5-10 ml Blood in adult culture bottle Children - 1-5 ml Blood in paediatric culture bottle Do not open cap. Clean cap with spirit and then inject blood into bottle by needle through hole in the cap. Do not refrigerate. In case of delay, store in a incubator at 37 C or at room temperature. Ideally blood cultures should be sent in parallel from two different sites (where possible) before starting antibiotic therapy. For blood culture by BACT Alert 3D, (For ICU, NICU, Paediatric cases, Nursery etc) ml) FA Plus-Adult (Green colored) add 5-10 ml Blood. PF Plus- Paediatrics (Yellow colored) 0.5 ml 4 ml Blood. (Minimum 1 6) URINE CULTURE Duly filled form should include clinical indications, details of antibiotics & diuretics administered h/o catheterization, renal stones, BPH, comorbities & previous culture reports. Mention sample collected from catheter/mid stream/supra pubic aspirate along with timing of collection early morning sample or not. Collect clean catch mid stream urine sample in sterile Universal container. Patient should be given instructions for collection of urine Transport to the laboratory immediately. In catheterized patients: Catheter is clamped above the distal end to collect freshly voided urine. Tubing is cleaned vigorously with 70% alcohol (spirit). Under all aseptic precautions, urine is aspirated with needle & syringe. Then, urine is collected in wide mouth sterile container. Never collect urine from bag. 7) PUS CULTURE AND SENSITIVITY Always mention source & site of sample taken Aspirated samples are preferred. Clean the wound area with sterile normal saline and collect the pus sample on a sterile swab stick (provided by the Microbiology Department) in sterile test tube following all aseptic precautions. Pus swabs should be visibly charged with pus, expressed pus samples preferred.
Send separate swab for staining purpose. Pus sample collected by I&D/FNAC should be directly transferred in a sterile container Do not collect PUS from dressing material Do not send sample in syringe with needle. Send separate form for ZN staining (For AFB). (Sufficient quantity of sample is a pre-requisite for ZN stain) 8) CSF CULTURE SENSITIVITY/CSF Antigen detection(bacterial and Fungal) Give relevant clinical history & details of CSF cytology if available. Collect 2-3 ml of CSF in sterile container under all aseptic precautions & transport immediately. In case of delay, store in a incubator at 37 C or at room temperature. Do not refrigerate. 9) STOOL a) CULTURE AND SENSITIVITY Collect the sample in sterile wide mouth container Send only a small amount (2-3 g). Sample should not be mixed with urine. Rectal swab can be collected in case of infants (Insert swab into the rectum and rotate gently). Swab should be visibly charged. b) MODIFIED ZN STAINING: For acid fast oocyst.(cryptosporidium, Isospora, Cyclospora etc) 10) STOOL FOR CHOLERA 1-2 ml stool specimen should be preferably sent in alkaline peptone water (transport media) provided by Microbiology department. Sample if collected in an ordinary container should be immediately transported to the Lab. 11) BODY FLUIDS AND ASPIRATES Mention date & duration of fever, site of infection, surgical procedure, blood counts, radiological findings & other relevant investigations done. Collect in sterile container and transport immediately. In case of delay, store in a incubator at 37 C or at room temperature. Do not use unsterile container regardless of investigation. 12) SPUTUM FOR AFB MICROSCOPY After rinsing mouth with plain water collect early morning coughed up sputum (not saliva) sample in clean wide mouthed container with screw tight fitting cover. A total of two consecutive samples are required to be submitted.
13) URINE FOR AFB Collect first early morning voided whole urine sample in clean, dry and sterile container. Send sample on at least three consecutive days. 14) MYCOBACTERIAL CULTURE Sputum, gastric aspirate, sterile body fluids, biopsies, pus, endometrial biopsy (pre menstrual) and first voided whole urine in sterile container. Blood is not an appropriate sample for mycobacterial culture. 15) CB NAAT For Mycobacterium tuberculosis Sputum, gastric aspirate, sterile body fluids and pus samples should be sent in sterile containers. Urine, blood & blood stained samples are not acceptable for CBNAAT. 16) ANAEROBIC CULTURE Aspirated pus samples in sterile containers. Tissue/biopsy in sterile containers. Swabs and surface samples are not acceptable. Samples should be transported to lab immediately after collection to maintain viability of anaerobes present in sample. Request forms must specifically mention anaerobic culture. 17) MYCOLOGY Give relevant clinical history including immune status of the patient & any preexisting disease. Area should be cleaned with 70% alcohol and let it dry. Skin, nail scraping / clipping should be sent in clean paper / sterile container Hair should be cut short & epilated with help of forceps along with hair root. Sputum: Coughed up, early morning 3 consecutive sputum samples in a sterile container after rinsing the mouth with plain water. CSF / Urine should be sent in sterile container. Sample should be delivered at the earliest. For corneal scraping, sterile Kit should be collected from department and immediately transferred to lab. Biopsy/tissue specimens should be sent in sterile saline. (NOT FORMALIN) 18) SEROLOGY / IMMUNOLOGY Without relevant clinical history required for the test, samples will not be processed.
Collect 3 ml venous blood (preferably fasting) in plain vial with aseptic technique. DO NOT COLLECT BLOOD FROM I.V. CATHETERS. Hemolysed samples will not be processed A. ASO, VDRL, TPHA, WIDAL, Brucella serology & Scrub typhus serology (3ml Blood sample in plain vial) B. CRP, PCT (3ml Blood sample in plain vial) C. C3, C4 (complement levels) / IgG, IgA, IgM, IgE (Immunoglobulin Profile) (3ml Blood sample in plain vial) Samples for immunoglobulin profile & complement levels should be sent from Monday to Wednesday only. D. For ELISA: Anti ccp, RF, ANA, APLA, DsDNA Collect 3 ml venous blood in plain vial under aseptic conditions E. Dengue & Chikungunya serology: Dengue (NS1 Ag / MAC ELISA / RAPID IgG, IgM), Chikungunya (IgM, MAC ELISA) Collect 2-3 ml blood in plain vial under aseptic condition. Haemolyzed sample shall not be accepted. All Dengue & Chikungunya requests to be accompanied by duly filled performa prescribed by NVBDCP (available on RMLH website. Platelet counts are must. F. ELISA for Hepatitis markers (HBsAg, Anti HBc IgM, Anti HBc total, HBeAg, Anti HBe, HCV Ag along with Anti HCV, Anti HAV & Anti HEV). Collect 4-5 ml venous blood in plain vial. Separate forms should be filled completely along with clinical details, duly signed and stamped, without which samples will not be accepted. Give clinical indication for the test along with LFT profile, h/o blood transfusion, and immunization for hepatitis B, h/o needle stick injury & any other injectables, should be mentioned in requisitions for Hepatitis B&C. G. ELISA for TORCH: Collection on 2 nd & 4 th Monday Collect 3 ml blood in plain vial. Give obstetric history, details of congenital anomalies in child, history of infertility & other clinical indications for the test. H. HIV:
Sample collected in Integrated Counselling and Testing Centre (ICTC Center in the Deptt. of Microbiology) after pre test counseling. Consent form is to be signed prior to the test. Collection time: 9.00 am to 11.30 am Monday to Friday, and 9:00 am to 11:00 am on Saturday. For voluntary clients and cases of needle stick injuries: 9.00 am to 3.00 pm Monday to Friday, and 9:00 am to 12:00 pm on Saturday. Reports are given by counselors after post test counselling (11:30 am to 4:00 pm) Note: All needle stick injuries to be reported to ICTC centre for counseling and testing. I. Leishmania serology: By rapid immunochromatographic test. 2-3 ml blood in plain/edta vial. J. Malaria & Filarial antigen: By rapid immunochromatographic test Collect 2-3 ml blood in EDTA vial. 19) CD4 counts and percentage: Test is done for only HIV positive patients. Samples collected from Monday to Friday (9:00 am to 11:30 am) in Deptt. of Microbiology. For indoor patients samples sent in vacutainers (EDTA vial) supplied by the Deptt. along with completely filled form and adequate clinical details. 20) MOLECULAR TESTS (PCR): A. TB PCR: following samples are acceptable in a sterile container- Sputum 2-5 ml (not saliva), preferably two samples. Gastric aspirate (2-5ml). Bronchoalveolar lavage BAL (2-5 ml). Urine CSF/ Aspirated body fluids Endometrial biopsy (premenstrual) Pus/Tissue/Biopsy/bone marrow Lymph node biopsy/aspirate Menstrual blood B. CMV PCR: Blood in EDTA vial (2-5 ml) Urine C. HIV, HCV, HBV PCR: Blood in EDTA vial (2-5 ml)
D. H1N1 PCR; Swab stick and viral transport media (VTM) are provided by laboratory. Collect nasopharyngeal swab and throat swab in VTM. Sample should be accompanied with specific PCR form available in microbiology laboratory. The request form must be signed by requesting faculty/consultant along with stamp. Request form must be accompanied with payment slip except for CGHS card holders, for thesis cases and in special cases if forwarded by respective head of departments and MS office. In case of BPL patients, sample can be requested for free processing from assigned authority. PCR for H1N1 is free of cost. Samples are to be deposited in microbiology laboratory at V1th floor PGIMER