Identification of Microorganism125577.pptx

Culture media

1. Identification of Microorganism Maxim T. Barikder

2. Questions Define culture & media. What do you mean by bacteriological culture media? Define culture medium. What do you mean by bacterial culture media? (DU-Jan 15, 13, 12) Classify media with example Classify culture media according to their constituents/ingredients. Classify bacterial culture media according to consistence. (DU-Jan 15, 13,12) Enlist the different culture media used in microbiological laboratory. Classify bacterial culture media based on consistency with example Write down the role of culture medium.

3. Questions Write down the importance of various media in disease diagnosis. Write down the role of media in identification of bacteria & fungus. What are the advantages and disadvantages of solid and liquid media? Describe the advantages of solid media over Write down the differences between solid & liquid media liquid media Write short note on: Selective media. Write short note on: Enriched media How can a media be enriched? (DU-13) Write down the difference between selective and enriched media. Why chocolate agar media is more enriched than blood agar media ?

4. Questions Write short note on: Indicator / Differential media. Write short note on: Transport media. Write down the methods/steps of culture/ isolation. Write short note on: Blood culture. Define blood culture? What are the indications of blood culture? (DU-Jan 16) Write down the indication of blood culture? (DU-Jan 15, 14) Mention 5 important indications of blood culture. What are the media used for blood culture? Enumerate different methods of blood culture .

5. Questions Name the methods of blood culture. (DU-Jan 16) Describe the procedure of blood culture. Describe the procedure of blood collection for culture in a laboratory. Write down the procedure of blood collection for blood culture. (DU-14) What are the indications of culture of urine? Explain the procedure of sample collection of urine for culture. (DU-Jan 15) What are the media used for stool culture? Explain the procedure sample collection of stool for culture. (DU-Jan 15) What are the indications of culture of throat swab or sputum? Explain the procedure of sample collection of throat swab for culture.

6. Questions Define staining methods? What is the importance of staining in diagnostic microbiology? Classify staining methods with example? Mention different types of staining used in the laboratory of microbiology Mention the common staining methods adapted for bacterial study Write down the principle of Gram staining. State the basic principle of Gram staining. Write down the steps of Gram staining. Define specimen? What do you by specimen?

7. Questions Define sample collection? What do you mean by specimen collection? Write down the importance of specimen collection. What are the purposes of sample collection? Write down the general principles of sample collection for microbiological laboratory examination. (DU Jan 17) Write down the general principles of sample collection for culture. (DU-Jan 16, 12 )

8. Definition Culture - It is the artificial process of cultivation of microbes by providing the proper environmental condition . Or Culture is the term given to microorganisms that are cultivated in the lab for the purpose of identifying and studying them . Media – It is an artificial food for the cultivation of bacteria in the laboratory . Or, Medium is the term given to the combination of ingredients that will support growth and cultivation of microorganisms by providing all the essential nutrients required for the growth . Example :. Mac Conkey's agar medium, Blood agar medium, Chocolate agar medium etc.

9. Criteria of good culture media Contains essential nutrients for the growth of the desired microorganism in proper concentration . Contains an adequate amount of salt . Contains an adequate amount of water . Must be sterile . Is of desired consistency . Must free from inhibitory substances for the organism to be cultured. Proper pH

10. Uses of Culture media 1. To identify the cause of infection from the clinical sample, so that proper treatment can be given. 2. To study the characteristics or properties of microorganisms. 3. To prepare biological products like vaccines, toxoids, antigens.. etc . 4. To see sensitivity to drugs like antibiotic & antifungal agents. 5. To transport certain bacteria.

11. Classification of Culture media On the basis of consistency Solid media; e g. nutrient agar, blood agar, chocolate agar etc . Semisolid media; c.g. soft agar media Liquid media; Peptone water, bile broth, sugar broth, Loeffler's scrum, Robertson's cooked meat media etc . B. On the basis of oxygen requirement ; Aerobic media . Anaerobic media . Eg . Thioglycolate broth, Robertson’s cooked meat broth.

12. Classification of Culture media C. On the basis of chemical / nutritional composition ; Simple / basal media; For general purpose. Example-nutrient broth, nutrient agar, peptone water, glucose broth etc . Special media ; Enriched media; e.g. blood agar medium, Lowenstein-Jensen medium . Enrichment media; e.g. Tryptic soya broth . Selective media; e.g. Mac Conkey's agar, blood tellurite agar.. Indicator / differential media; e.g. blood agar, Mac Conkey's agar. Transport media; e.g. Stuart medium, Carry-Blair medium . Storage media; e.g. egg-saline medium.

13. Advantages of solid media Bacteria may be identified by studying the colony character. Mixed bacteria can be separated. Discrete growth as colony. Easy to study morphology of bacteria Easy to pick-up . Easy to handle. Isolation of bacteria as pure culture is possible.

14. Disadvantages of solid media Need large number of bacteria in the specimen . Biochemical test cannot be done. Large inoculation cannot be tested.

15. Advantages of liquid media Used for profuse growth, as rapid multiplication of bacteria occur. So, small amount of specimen is sufficient. Biochemical tests can be done easily. Large inoculation can be tested Useful enrichment media Helps in transport of a specimen

16. Disadvantages of liquid media Mixed organisms cannot be separated. No colony formation. Identification of bacteria is not possible. Isolation of bacteria as pure culture is not possible.

17. Enriched media Substances like blood, serum, egg are added to the simple medium . Used to grow bacteria that are exacting in their nutritional needs. Enriched media is liquid in nature. Example- Blood agar- Streptococcus species Chocolate agar-Pneumococcus, Neisseria, Haemophilus . Lowenstein-Jensen medium- Mycobacterium tuberculosis.

18. Enriched media

19. Enrichment media Enrichment media refers to the liquid media that inhibits the growth of unwanted bacteria. Enriched media allow the growth of a wide variety of microorganisms. Enrichment media allow the growth of a particular type of microorganism in the medium . e.g. Tryptic soya broth.

20. Selective media The inhibitory substance is added to a solid media to inhibit commensal or contaminating bacteria such as: Antibiotics Dyes Chemicals Alteration of pH Altering temperature Removal of O2 Selective media is solid in nature. Eg . Mac Conkey's agar, blood tellurite agar, TTGA (Tellurite Taurocholate Gelatine agar) or, Monsur's media, Deoxycholate citrate agar(DCA)

21. Differential media Differential media are designed in such a way that different bacteria can be recognized on the basis of their colony color. Dyes and metabolic substrates are incorporated so that those bacteria that utilize them appear as differently colored colonies. Examples: Mac Conkey's agar , CLED agar, TCBS agar, XLD agar

22. Mac Conkey's agar

23. Transport media Media used for transporting the samples. Delicate organisms may not survive the time taken for transporting the specimen without a transport media. Example Cary-Blair medium-for salmonella. Shigella , Vibrio . Amies medium -For gonococci & other pathogens. Monsur's medium-For Vibrio cholera Stuart's transport media for gonococci . Alkaline peptone water - For Vibrio

24. Stapes of Culture Collection of specimen in a sterile container . Transport or storage without killing microorganisms . Inoculation in appropriate medium . Incubation of the inoculated medium at optimum temperature, in correct atmosphere for an adequate period.

25. Basic requirements for culture in a laboratory 1 ) Specimen . 2 ) Appropriate media . 3 ) Bacteriological loop . 4 ) Spirit lamp . 5 ) Biological safety cabinet class I to IV (if available ). 6 ) Incubator

26. Basic constituents of basal media 1 . Water . 2 . Salt - NaCl . 3 . Peptone: a mixture of polypeptide & amino acids . 4 . Meat extract, yeast extract.

27. Colony & Incubation Colony is the macroscopic mass of bacteria produced by multiplication from a single bacterium in the proper environmental condition . Incubation is provision of proper condition for the growth and development of organisms within the culture media. For most of the bacteria optimum incubation temperature is 37 ° Colony count is a measurement of the growth of bacteria in a sample that has been cultured. The standard plate count method is an indirect measurement of cell density and reveals information related only to live bacteria.

28. Bacteria which can not be cultured Treponema pallidum Mycobacterium leprae Chlamydiae

29. Blood Culture

30. Blood Culture Blood culture is a test designed to detect if microorganisms such as bacteria and fungi are present in blood. A sample of blood obtained using sterile technique is placed in a culture media and incubated in a controlled environment for 1 to 7 days . Or A blood culture is a laboratory test to check for bacteria or other germs in a blood sample.

31. I ndications for B lood C ulture Enteric fever . Septicemia. Bacteremia Meningitis. Pyrexia of unknown origin Bacterial infective endocarditis Osteomyelitis. Brucellosis. Urinary tract infection (If there is fever, rigors or evidence of septic shock).

32. Blood Culture Media The following media are used to culture a blood sample Tryptic soya ( Tryptone soya, trypticase soya) broth . Diphasic tryptone soya medium . Bile salt broth with or without Gention violet . Thioglycolate broth for growth of anaerobes . Brain heart infusion broth . Subculture done in: Blood agar media, Mac Conkey's agar media, Chocolate agar media.

33. Methods of Blood Culture Blood culture can be done in three processes – Traditional method - Using liquid media on biphasic media Lytic method - After processing in lytic solution in solid media Automated - very rapid (within 6 hour)

34. Articles Required for Blood C ulture Tourniquet . Small mackintosh . Syringes : 3ml 5ml, 10ml . No 20 Gauge needles . Alcohol swabs . Disposable gloves . Specimen container- test tube or bottle . Laboratory requisition form Sterile gauze pads ( 2’’ X2’’ ) Adhesive tap

35. Procedure Check the physician's order, progress, notes, and nursing care plan Identify the patient. Explain the procedure to the patient. Collect and prepare equipment. Ensure patient's privacy . Wash and dry hands. Put on clean, disposable gloves. To avoid hemolysis, the needle used for venipuncture should have a short berel , the syringe and needle must be dry, and the container into which the specimen is placed must be dry. For some tests whole, clotted blood is required, whilst for others it should be anti coagulated, so that the laboratory may obtain the fraction of the blood for examination that they require

36. Procedure Each laboratory should issue specimen bottles containing the correct anticoagulant for each test, with instructions as to the quantity of blood required. Heparin , potassium oxalate and sodium citrate are fairly common anticoagulants. For blood sugar estimation fluoride is added to the tube to prevent the disappearance of the sugar from the blood. Once the blood has been added to the anti-coagulant, it must be inverted gently so that it is thoroughly mixed with the specimen. When sending blood for examination, care must be taken to avoid hemolysis of the specimen , for hemolysis renders the specimen useless. Shaking causes hemolysis. Document the procedure appropriately

37. Standard collection protocol Collect up to 3-4 sets of blood culture bottles. Two blood culture bottles in each set . Obtain 5 ml blood per bottle. Draw from up to 3 different venipuncture sites . Space blood collections by 1 hour intervals.

38. Blood Collection Tube

39. Urine Culture

40. Urine Culture   Urine culture is  a lab test to check for bacteria or other germs in a urine sample . It can be used to check for a urinary tract infection in adults and children

41. Indication of Culture of Urine To diagnose the following diseases- Urinary tract infection (UTI ). Enteric fever/ typhoid fever . PUO (pyrexia of unknown origin ). AGN (acute glomerulonephritis )- inflammation and subsequent damage of the glomeruli leading to hematuria(Blood in urine), proteinuria(Protein in urine), and azotemia( Elevated BUN)

42. Equipment for Urine Culture Clean, wide mouthed container . Bed pan or urinal. Soap and water. Appropriate laboratory form. Gloves. Laboratory requisition form

43. Urine Collection Container

44. Bed pan

45. Urinal

46. Procedure for Urine Culture Check the physician order and nursing care plan . Identify the patient Explain procedure to the patient with specific instructions about washing the genital area (skin around the urethra meatus) with soap and water and give the labeled container. Instruct patient not wet the label on the outside. Ask the patient to direct the first and last part of the urine stream into a urinal or toilet and to collect the middle part of the stream into the special container.

47. Procedure for Urine Culture Have the patient place the specimen container in proper / designated place. With gloved hand place the specimen container in polythene bag. Send specimen to the laboratory with completed, signed laboratory form. Remove gloves and wash hands . Record the procedure in the nurse's notes and other appropriate forms .

48. Stool Culture

49. Stool Culture The stool culture is a test that detects and identifies bacteria that cause infections of the lower digestive tract.

50. Indications for Stool Culture Bloody stool Occult blood(hidden blood) or leukocytes P rolonged course of diarrhea

51. Culture Media for Stool Culture Mac Conkey's agar media . XLD (Xylose Lysine Deoxycholate ). DCA ( Deoxycholate Citrate agar ). Tetrathionate broth (Selenite F broth).

52. Equipment for Stool Culture A clean specimen container . A spatula for putting the specimen into the container . Dry bed pan (for helpless patients ). Additional bedpan for rinsing and cleaning . Laboratory requisition form . Clean gloves . Waste paper (for wrapping used spatula ). A pitcher of water (for helpless patient ). Tissue/towel .

53. Specimen container for stool

54. Procedure for Stool Culture Check the physicians order. Identify the patient . Explain to patient the procedure and make clear what is expected of him/her. Give the labeled container and spatula to the patient with instructions-To defecate into clean bedpan & not to contaminate specimen with urine . Don gloves . For helpless patient: assist patient on to the clean bedpan.

55. Procedure for Stool Culture Leave him with instructions. When done, remove and keep aside the bedpan after placing the second one for cleansing. Once the specimen is collected sent it to lab with the appropriate requisition forms. Wash and replace the reusable articles. Dispose of the used spatula wrapped in waste paper. Wash and dry hands. Record information in the patient's chart.

56. Sputum/Throat Swab Culture

57. Sputum/Throat Swab Culture A throat swab culture is a laboratory test that is done to identify germs that may cause infection in the throat . A sputum culture is a test that checks for bacteria or another type of organism that may be causing an infection in your lungs or the airways leading to the lungs.

58. Indication for Sputum/Throat Swab Culture Diphtheria Streptococcal sore throat (tonsillitis, pharyngitis ) Tuberculosis Pneumonia. Bronchitis Pulmonary abscess

59. Articles for Sputum/Throat Swab Culture Sterile cotton-tipped applicator specimen collection Tongue depressor Laboratory request form Flashlight

60. Sterile cotton-tipped applicator specimen collection

61. Tongue depressor

62. Procedure for Sputum/Throat Swab Culture Always observe proper hand hygiene prior to the test . Have the patient sit comfortably either on bed or chair while explaining the procedure. Allow the patient to tilt his head back. Make use of the flashlight to light up the back of the throat and check for presence of inflammation using the tongue depressor . Swab the tonsillar areas from side to side and make sure to include any inflamed purulent sites. The test may cause momentary gagging because the back of the throat is a sensitive area, but it should not be painful.

63. Procedure for Sputum/Throat Swab Culture Refrain from touching the tongue, cheeks, or teeth with the applicator, due to possible contamination with oral bacteria. Place the cotton-tipped applicator into the culture tube immediately. Label the culture tube with the patient's name, SSN, and ward number if applicable. Fill out the request form completely The sample is then taken to the laboratory for culture.

64. Staining Methods

65. Staining Methods Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes . STAIN - Stain is a dye used to color the living or dead organelles.

66. Importance of Staining in Diagnostic Microbiology Identification of different bacterial species . To know about the details morphology of the bacteria by different types of staining. To select proper antibiotics, as we know there are wide variation of antibiotic sensitivity between Gram positive, Gram negative & acid-fast For example- Gram positive bacteria are more sensitive to ß-lactam antibiotics than Gram-negative bacteria. So, identification of bacteria whether it is Gram positive or Gram negative is important prior to start treatment.

67. Classification of Staining Used in Microbiology POSITIVE STAINING:- where the actual cells are themselves colored and appear in a clear background. (a) Simple staining: A stain which provides color contrast but gives same color to all bacteria and cells. Ex : Loeffler's methylene blue, Polychrome methylene blue, Diluted carbol fuchsin (b) Differential Staining: A stain which imparts different colors to different bacteria is called differential stain(which contains more than one stain ). Ex : Gram's stain, Acid fast staining, Special stains.

68. Classification of Staining Used in Microbiology NEGATIVE STAINING: where the cells remain clear (uncolored) and the background is colored to create a contrast to aid in the better visualization of the image.eg . Indian ink & Nigrosin Fluorescent stains : Example: Auramine and rhodamine to detect mycobacteria. Impregnation stain :Example : Silver impregnation for Treponema pelidum .

69. Principals of Gram Staining Gram staining depends on the structures of bacterial cell wall & cytoplasmic membrane. Gram-positive bacteria have thick peptidoglycan layer on their cell wall, so they can resist decolorizing of primary stain by alcohol or acetone mixture . Gram positive bacteria can not be stained by a counter stain. Gram-negative bacteria have a thin peptidoglycan layer, so they cannot prevent decolorizing by alcohol or acetone mixture, and so they are stained with a counter stain.

70. Smear Smear is a distribution of bacterial cells on a slide for the purpose of viewing them under the microscope. Method: Aseptically a small sample of the culture is spread over a slide surface. This is then allowed to air dry. The next step is heat fixation to help the cells adhere to the slide surface. The smear is now ready for staining

71. Smear

72. PROCEDURE Make a thin smear on a slide Heat fix the smear by passing the slide 23 times gently over the Bunsen flame with the smear side up Pour Loeffier's methylene blue over the smear and allow it to stand for 3 minutes . Wash the stained smear with water and air dry it Observe the smear first under low power (10) objective, and then under oil immersion (100x) objective Observe the presence of organisms and also the cellular content of sample.

73. Steps in Gram staining 1. Preparation & fixation of smear: At first clean a slide and make a thin film with supplied specimen. Then dry it in the air. Fix the film by slowly passing the slide 3-4 the times through a flame . 2 . Primary staining: Cover the film with crystal violet and kept for 1 minute. It is then washed with tap water. 3. Mordanting : The slide is covered with a solution of Gram's iodine/ Lugol's iodine for 1-2 minutes 4 . Decolorizing: It is done by acetone or alcohol and is continued till the violet-color comes out from the smear. The slide then washed with tap water . 5 . Counter staining: Cover the slide with dilute Carbol Fuchsin for 20-30 seconds or 5 drops of Safranine for one minute . Wash with water and dry in air.

74. Findings of gram staining Cells that retains the color of Primary Stain( Purple) are Gram positive . Calls that do not retains the color of Primary Stain and takes up the color of Counter Stain ( pink or red ) are Gram Negative.

75. Sample / Specimen collection

76. Sample / Specimen collection A specimen may be defined as a small quality of a substance or which shows the kind and quality of the sample . or A specimen of anything that is selected for diagnosis examination study or testing . or A specimen is a small quantity of a substance which is collected for investigation purposes and this sample shows the kind and quality of the whole substances. Specimen collection is the collection of a required amount of tissue or fluid for laboratory examination is called specimen collection. Or The collection of tissue or body fluids for laboratory investigations to aid diagnosis and/or treatment of medical conditions.

77. Purpose/Importance of Specimen Collection: To collect a specimen of body discharges or excreta for examination. To aid the doctor is diagnosing and treating the disease. To detect any abnormalities. To identify the disease and nature of disease. To determine the cause for treatment. To note the progress of disease.

78. Principals of specimen collection The patient should be informed in advance regarding the procedure and sample of specimen. The specimen container should be clean. Every specimen should be separately labeled clearly with patient's name, bed number, reg. number, ward number, etc. and nature of specimen with nurse's signature and date or time of collection etc . All specimens duly labeled should be kept in separate tray for dispatching to lab. Morning specimens should be collected by the night duty nurse on duty. Other samples should be collected by day nurse only . Any special instruction for special test should be noted by nurse in nursing lab note book and instructions is followed accordingly.

79. Types of Specimen A. Routine examination Urine for R/M/E . Stool for R/M/E . Blood for TC, DC, ESR, Hb %. B. Special examination : Excretion/Body secretion Urine, Stool, Blood, Sputum, Wound discharge, Vaginal swab, Throat swab, Nasal swab . Aspiration: Ceribral spinal fluid, Peritoneal fluid . Biopsy: Segment of the body tissue.