Quality control in microbiology

A brief yet informative presentation of the dos and donts of Microbiology lab.

1. QUALITY CONTROL IN MICROBIOLOGY PRESENTER:DR TABEEN MANSOOR 1 st year PG MICROBIOLOGY Sheri Kashmir Institute Of Medical Sciences

2. WHAT IS QUALITY? Quality means meeting the pre-determined requirements of users for a particular substance or service . Quality includes the following : Total Quality Management (TQM) Continuous Quality Improvement (CQI) Quality Assurance (QA)

3. QUALITY ASSURANCE Quality assurance has been defined by WHO as: “The total process whereby the quality of laboratory reports can be guaranteed .”

4. QUALITY ASSURANCE

5. QUALITY CONTROL(QC) The term QC covers that part of QA, which primarily concerns the control of errors in the performance of tests and verification of test results. QC must cover all aspects of every procedure within the department . The laboratory director is responsible for both QA& QC.

6. QUALITY CONTROL AND ASSURANCE QC is associated with internal activities that ensure diagnostic test accuracy. QA is associated with the external activities that ensure positive patient outcomes. Reduced length of stay Reduced cost of stay Reduced turn around time for diagnosis of infection Change to appropriate antimicrobial therapy Customer (physician or patient satisfaction)

7. THE DIAGNOSTIC CYCLE

8. PRE ANALYTIC PHASE

9. SPECIMEN COLLECTION SITE OF COLLECTION Must be from the actual site Minimum contamination from adjacent tissues, organs or secretions Swabs are inferior in the collection of most specimens Use of aspiration needles and catheters should be encouraged A patient information sheet should be given to patients for collection of urine specimens.

10. SPECIMEN COLLECTION TIME OF COLLECTION Optimal time of collection. Pathophysiology of the infectious disease should be known Blood cultures are usually positive in the first week Urine and stool culture positive during 2 nd and 3 rd week of illness 24 hours collection of sputum and urine should not be done.

11. SPECIMEN COLLECTION QUANITY OF THE SPECIMEN Must be sufficient Guidelines should be established to define a sufficient volume. If quantity is low Tubes containing holding broth such as physiologic saline(non nuntrient ) or phosphate yeast glucose (PYG) should be provided.

12. SPECIMEN COLLECTION DEVICE FOR COLLECTION Sterile containers should be used. Wide mouthed T ightly fitted caps to prevent leakage and contamination Swabs tipped with Dacron or Rayon polyester are better choices.

13. SPECIMEN COLLECTION Specimen should not remain in contact with swab for long duration. Swabs should be placed in transport media to prevent drying ( upto 48 hours). EXCEPTIONS : Skin scrapings and nail clipping for recovery of dermatophytes should be submitted dry in a clean container Prevents overgrowth of bacteria. Swab for recovery of Streptococcus pyogenes Bacteria that colonize dry off.

14. SPECIMEN COLLECTION ASPIRATE YIELDED A PURE CULTURE OF STAPHYLOCOCCUS AUREUS.STAPH WAS ALSO RECOVERED IN THE SPECIMEN BY SWABBING BUT IT WAS MIXED WITH OTHER CONTAMINATING FLORA

15. SPECIMEN COLLECTION SPECIMEN BEFORE ANTIBIOTIC ADMINISTRATION Obtain cultures before administering antibiotics Ideal for organisms highly susceptible to antibiotics B-haemolytic streptococci from throat specimens Neisseria gonorrhoea from genitourinary samples H.influenza or Neisseria meningitis from CSF

16. SPECIMEN COLLECTION SMEARS SHOULD BE PERFORMED Provide clues to the inflammatory nature of the condition. Indicates whether the result of culture are meaningful or not A wound swab which contains no PMNs but yields mixed bacterial growth cannot be considered valid

17. SPECIMEN COLLECTION PROPER LABELLING Identification number Name/Age and gender Source of specimen Clinician in charge Date/Hour collected Diagnosis Any antibiotics given Investigation required

18. SPECIMEN TRANSPORT Primary objective is to maintain sample in its original state. Adverse environmental conditions such as Extremes of heat and cold Rapid changes in pressure(during air transport) Excessive drying s hould be avoided For prolonged delay (>4 days) specimens should be frozen at -70 deg . Samples for recovery of mycobacteria and fungi should be shipped immediately

19. SPECIMEN RECEIPT & PRELIMINARY OBSERVATION A rea should be designated for receipt of specimens. Initial observation and handling in the biosafety cabinet. Personnel should wear protective clothing Lab Coats Rubber gloves and In some cases custom fitted masks

20. SPECIMEN RECEIPT & PRELIMINARY OBSERVATION 1.ENTRY OF DATA Essential data should be entered into a log book or computer data base. 2.GROSS EXAMINATION Visual examination & determination whether all criteria for acceptance are met. 3.MICROSCOPIC EXAMINATION D irect mounts or stained smears to establish a presumptive diagnosis

21. CRITERIA FOR SPECIMEN REJECTION Any specimen received in formalin 24 hour sputum collection Smears of secretions from uterine cervix, vaginal canal or anus for Gram’s stain detection of Neisseria gonorrhoea. A single swab submitted for multiple requests Eg : aerobes,anaerobes,fungus and tuberculosis Improper collection site like stool for respiratory syncytial virus

22. CRITERIA FOR SPECIMEN REJECTION Leaking container Unlabelled/wrongly labelled/mismatched samples Quantity Not Sufficient For Testing (QNS) Prolonged transport Dried specimen Haemolysed blood Lipemic serum Wrong tube

23. SPECIMENS UNSUITABLE FOR ANAEROBIC CULTURE: Gastric washings Midstream urine Prostatic secretions collected transurethrally Faeces(except for recovery of C.difficle, C.perfringens, C.septicum) Ileostomy or colonostomy swabs Throat ,nose or other oropharyngeal specimens(except specimens obtained from deep tissue during oral surgery) Superficial skin and environmental cultures

24. ANALYTIC PHASE

25. MICROSCOPIC EXAMINATION 1.Number and percentage of segmented neutrophils Indicate the magnitude and type of inflammatory response. Quality of specimen can be validated 2.Observation of bacteria, mycelial elements, yeast gives an immediate presumptive diagnosis 3.Presumptive evidence that species of anaerobic bacteria are present.

26. QC OF STAINS All stains and reagents must be clearly labelled, dated , and stored correctly. Should not be used beyond their expiry date Should not be used when they show signs of deterioration like abnormal turbidity and decolouration . At regular intervals and whenever a new stain is prepared, control smears should be stained Smear should not be too thick. D ecolourization is often incomplete which can result in gram negative organisms being reported as gram positive

27. QC OF STAINS

28. PROCESSING THE SPECIMEN 1.Select the primary culture media for the specimen 2.Select the temperature and conditions for incubation. 3.Determine which of the following isolates recovered on primary media require further characterization 4.Determine whether antimicrobial susceptibility tests are required

29. PROCESSING SPECIMENS Panculture - Indiscriminate ordering of cultures from all accessible body sites in hope of recovering a pathogen should not be done Non selective media free of inhibitors support the growth of most bacteria 5% sheep blood agar is the most commonly used Chocolate agar should be used where Haemophilus and Neisseria species are suspected ie.in sputum, CSF and semen.

30. PROCESSING SPECIMENS PRIMARY CULTURE MEDIA Inhibitory agars should not be used alone May inhibit use of organisms of interest. Broth cultures are done in special situations like Spontaneous bacterial peritonitis Peritoneal infections in patients of peritoneal dialysis Septic arthritis

31. PROCESSING SPECIMENS TRANSFERING SPECIMEN ON A CULTURE PLATE: Should be carried out in a biosafety cabinet Rubber gloves should be worn Appropriate charts and instructions posted on a bulletin board Or included in the bench manual for those who are new to the lab.

32. SEEDING A CULTURE PLATE E ssential to learn the skills of inoculating specimens. Instrument for seeding media selected according to the nature of the medium and inoculum. Platinum or nichrome wires of different gauges are used. Nichrome has oxidizing properties T ests where this property of bacterium is to be tested (e.g. oxidase test), platinum wire, instead of nichrome should be used.

33. SEEDING A CULTURE PLATE This wire is sterilized by holding it vertically in the flame of the burner so that the whole length of wire becomes red hot. It is allowed to cool down before it touches any material suspected to be having bacteria to avoid the heat killing the organisms. Pre sterilized disposable loops are now available commercially . The wire can be used as a: STRAIGHT WIRE T o stab the culture P icking of single colonies I noculating the liquid media

34. SEEDING A LIQUID MEDIUM THICK WIRE /LOOP F or lifting viscid material such as sputum S eed a plate of medium as the straight wire usually cuts the agar FOR LIQUID MEDIA If the tubes have got cotton plugs The mouth of the tubes should be heated in flame before and after any handling of tube N ot required when metal caps and screw-capped tubes are handled

35. PROCESSING SPECIMEN

36. SEEDING A MEDIUM SUBCULTURE FROM A SOLID MEDIUM TO SOLID MEDIUM Using a sterile wire or loop, a representative colony is touched and sub cultured onto appropriate solid medium by touching the wire or loop onto SURFACE OF THE MEDIA ➢ When more than one medium is inoculated, follow a particular order. Inoculate media without inhibitors , followed by indicator and then selective media . ➢ While processing fluid specimen inoculate liquid media first to reduce the chances of carry over from contaminated solid media.

37. ASEPTIC TECHNIQUES Open caps and lids of containers for the briefest period. Do not keep lids on the workbench. Inoculating loops should be put through the flame While working on the infectious material, keep the specimen away from the face .

38. ASEPTIC TECHNIQUES Loops should not contain fluid or large particles of matter that may splatter when placed in the flame. Avoid vigorous shaking of the specimen prior to opening. O pen the caps slowly to minimize aerosol production. H omogenization & grinding of tissue or biopsy specimen should be done in safety cabinet .

39. ASEPTIC TECHNIQUES Keep all specimens in racks to reduce the risk of accidental spillage . Mop up the workbench clean with disinfectant at the start and close of work . Wash hands with soap and water before and after handling infectious specimens

40. INTERPRETATION OF CULTURES Interpretation of primary cultures should be done after 24-48 hrs R equires considerable skill. By assessing the colonial characteristics the microbiologist can make a preliminary identification of bacteria. This is one of the cornerstones of diagnostic microbiology.

41. PRELIMINARY TESTS & THEIR QC

42. ANTIMICROBIAL SUSCEPTIBILITY TESTING Salient features of quality assurance in antibiotic susceptibility testing Use antibiotic discs of 6 mm diameter. Use correct content of antimicrobial agent per disc . Stock the supply of antimicrobial discs at - 20 Deg C . Use Mueller-Hinton medium for antibiotic susceptibility testing.

43. ANTIMICROBIAL SUSCEPTIBILITY TESTING Use appropriate control cultures. Use standard methodology for the test . Use coded strains from time to time for internal quality control .

44. ANTIMICROBIAL SUSCEPTIBILITY TESTING Space the antibiotic discs properly to avoid overlapping of inhibition zone . Use inoculum size that produces near confluent growth. Ensure an even contact of the antibiotic disc with the inoculated medium . Measure the zone sizes precisely. Interpret the zone sizes by referring to standard charts . Keep the antibiotic discs at room temperature for one hour before use. Incubate the sensitivity plates for 16-18 hours before reporting. Incubate the sensitivity plates at 35 deg C.

45. QC OF EQUIPMENTS

46. QC OF EQUIPMENTS

47. QC OF EQUIPMENTS

48. QC OF CULTURE MEDIA Water used for the media should be free of Copper ions The pH of the water should be slightly on the acidic side, but should not be less than 5.5. The conductivity should be less than 15 µS ( microsiemens ). Media prepared should be free from excessive bubbles or pits. Should be free of cracks, freezing or crystallization. Thickness of the medium should be 4mm Should be checked at four points being at right angles to each other Only borosilicate glassware should be used because soda glass can leach alkali into the media.

49. QC OF CULTURE MEDIA

50. MICROBIOLOGICAL QC OF MEDIA The control organism is inoculated in soyabean casein digest (SCD) broth I ncubated for 4 hours to get a cell density comparable to 0.5 McFarland’s standards . S tandard suspension should give a colony count of 107- 108 cfu /mL (0.08-0.1 absorbance at 625nm). A 10 μL quantity of inoculum of 1in 10 and 1 in 100 dilution in normal saline or in SCD broth should be used for selective and nonselective

51. MICROBIOLOGICAL QC OF MEDIA These diluted inocula are used to ascertain the growth supporting capacity of the media. The inoculation is done in duplicates for each type of inoculum. After inoculation, the plates are incubated at 37°C for 24 hours T heir growth and colony characteristics are observed. The results can be reported by mentioning presence or absence of growth and the growth characteristics in a tabular form as shown in Table 1.

52. COST EFFECTIVENESS RULE OF THREE: As a guideline Schreckenberger and Miller have suggested the “rule of three” 1 or 2 potential pathogens should be evaluated even in the presence of commensal flora BUT 3 or more pathogens should not be evaluated

53. POST ANALYTIC

54. REPORTING OF RESULTS Should be quick Terminology should be understandable Reports maybe designated as Urgent or important Urgent reports must always be telephoned to the caregiver Confidentiality of the patient data must be ensured Timely ,preliminary and interim reports should be issued Reference range should be given where appropriate. To avoid time and labour reports that are negative should be stamped A copy of the report dispatched should be kept in the lab

55. INTERACTION WITH EPIDEMIOLOGIST Certain infectious agents must be reported to the public health authorities. They are different for different regions. Microbiologists should remain alert for unusual patterns of isolates.

56. MAINTENANCE OF SAMPLES & RECORDS Local and National guidelines must be followed. All patient records should be maintained for atleast 2 years and ideally for 10 years . Sterile body fluids should be maintained at room temp until culture and other procedures have been performed Blood culture isolates should be maintained for 30 days. All positive cultures should be kept for 7 days for further evaluation like molecular typing.

57. MAINTENANCE OF SAMPLES & RECORDS Viruses maybe frozen to -70 deg in a solution containing a cryoprotectant like 10% dimethyl sulphoxide (DMSO). Tissue should be frozen at -70 deg for potential future use. CSF should be stored at room temp because of lability of Neisseria meningitides at 4 deg C

58. MAINTENANCE OF SAMPLES & RECORDS Non fastidious aerobic bacteria can be saved upto 1 year on TSA slants Long-term storage of aerobes and anaerobes can be accomplished by lyophilisation (freeze drying) or freezing at -70 deg. Frozen, non fastidious organisms should be thawed, re isolated and refrozen every 3 years. Stock isolates may be maintained by freezing them in :-- 10% skim milk TSB with 15% glycerol 10 % horse blood in sterile vials

59. MAINTENANCE OF SAMPLES & RECORDS Yeasts should be stored like non fastidious bacteria Moulds can be stored on potato dextrose agar (PDA) slants at 4 deg for 6 months -1 year Long term storage in PDA slants overlaid with sterile mineral oil at room temp Fungi can also be maintained as water cultures at room temperature. AFB may be kept on LJ agar slants at 4 deg for 1 year. Or frozen at -70 deg in 7H9 broth with glycerol

60. STANDARD OPERATING PROCEDURE(SOP) Essential component of QC Should be written in format of CLSI guidelines. Must be reviewed and signed annually or biannually by the laboratory director FUNCTION I mprove and maintain the quality of laboratory service to patients I dentify problems associated with poor work performance. T o provide laboratory staff with written instructions on how to perform tests consistently to an acceptable standard in the laboratory . T o help avoid short-cuts being taken when performing tests. To provide safety in laboratory

61. STANDARD OPERATING PROCEDURE(SOP) I nfrastructure of a laboratory Biosafety precautions Disposal of infectious waste Collection , transport and storage of specimens Reagent preparation Criteria of rejection of samples Processing of specimens Maintenance of equipment Recording of results Reporting of results Tolerance of limits Procedure of quality control Referral Each laboratory should have Standard Operating Procedure Manuals (SOPMs) which should include the following information about the

62. FACILITIES IN A LABORATORY Each laboratory must possess space for : S ample collection S ample analysis S torage of samples R eagents , chemicals Stationary& record etc .

63. FACILITIES IN THE LABORATORY Washing Media preparation Autoclaving S eminar room L ibrary S taff room and Toilets

64. FACILITIES IN THE LABORATORY Laboratory must be well lit with dust-free Air conditioned environment. U ninterrupted power supply. The laboratory must monitor, control and record environmental conditions B iological sterility Humidity T emperature .

65. FACTORS IN PREVENTING THE ESTABLISHMENT OF ESSENTIAL MICROBIOLOGY SERVICES IN DEVELOPING COUNTRIES High cost of culture media and reagents L ack of rational approach to the selection and use of microbiological investigations S hortage of trained technical staff Shortage of clinical microbiologists.

66. STAFF QUALIFICATIONS SUPERVISORY PANEL: Small and medium laboratory May be manned by an MBBS or an MSc in concerned speciality with at least 5 years experience in laboratory medicine. Large and superspeciality laboratory: Shall be manned by a medical person with postgraduate qualification in Microbiology in their respective division.

67. STAFF QUALIFICATIONS TECHNICAL PERSONAL 1 technologist per 500-1000 specimens per year. S hould have one of the following qualifications : a) Graduate in medical laboratory technology. b ) Science graduate with one-year experience in a medium-sized laboratory. c ) Diploma in medical laboratory technology with 2 years experience in a medium-sized laboratory. d ) A laboratory may appoint up to 25% of staff without experience but with requisite qualifications or with more than 10 years of laboratory experience with at least matriculation in science.