Staining & culture medias


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Staining & Culture Medias

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Staining & culture medias

  1. 1. Abbas Naseem (BDS) Dated: Nov 7, 2010
  2. 2. Chocolate agar Chocolate agar (CHOC) - is a non-selective, enriched growth medium. It is a variant of the blood agar plate. It contains red blood cells, which have been lysed by heating very slowly to 56 °C. Chocolate agar is used for growing fastidious (fussy) respiratory bacteria, such as Haemophilus influenzae. These bacteria need growth factors, like NAD and hematin, which are inside erythrocytes; thus, a prerequisite to growth is lysis of the red blood cells 2
  3. 3. Blood Agar• A nutrient culture medium that is enriched with whole blood and used for the growth of certain strains of bacteria.• Contains mammalian blood (usually sheep or horse), typically at a concentration of 5–10%.• BAP are an enriched, differential media used to isolate fastidious organisms and detect hemolytic activity.• β-hemolytic activity will show complete lysis of red blood cells surrounding colony,• while α-hemolysis will only partially lyse hemoglobin and will appear green.• γ-hemolysis (or non-hemolytic) is the term referring to a lack of hemolytic activity. Different types of hemolysis on Blood Agar 3
  4. 4.  Cysteine Lactose Electrolyte Deficient agar (CLED) ◦ CLED agar is used to isolate and differentiate urinary tract bacteria, since it inhibits Proteus species swarming and can differentiate between lactose fermenters and non-fermenters. 4
  5. 5. MacConkey agar (MAC)  A selective and differential media used to differentiate between Gram negative bacteria while inhibiting the growth of Gram positive bacteria.  The addition of bile salts and crystal violet to the agar inhibits the growth of most Gram positive bacteria, making MacConkey agar selective.  Lactose and neutral red are added to differentiate the lactose fermenters, which form pink colonies, from lactose nonfermenters that form clear colonies. Composition: Bile salts Crystal voilet Lactose Neutral red• MacConkey agar is useful medium for cultivation of enterobacteria• It contains a bile salt to inhibit non intestinal bacteria• Lactose in combination with Neutral red distinguish the lactose fermenting from the non lactose fermenting Salmonella and Dysentery group 5
  6. 6. Lactose fermenting NON Lactose fermenting
  7. 7. Triple Sugar Iron medium• Triple Sugar Iron medium is a differential medium that can distinguish between a number of Gram-negative enteric bacteria based on their physiological ability (or lack thereof) to:• a. metabolize lactose and/or sucrose b. conduct fermentation to produce acid c. produce gas during fermentation d. generate H2S.• The medium contains 1.0% each of sucrose and lactose and 0.1% glucose.• If only glucose is fermented, acid produced in the butt will turn it yellow, but insufficient acid products are formed to affect the methyl red in the slant.• However, if either sucrose or lactose are fermented, sufficient fermentation products will be formed to turn both the butt and the slant yellow.• If gas is formed during the fermentation, it will show in the butt either as bubbles or as cracking of the agar.• If no fermentation occurs (as for an obligate aerobe), the slant and butt will remain red. 7
  8. 8. A 8-year-old boy developed sore throat and fever. On examination,grayish exudates (pseudomembrane) is found over tonsils andpharynx. The causative agent is grown on a medium whichproduces blackish colonies as show in the photograph.A. Identify the culture medium and the most likely the causativeorganism.B. Why the causative agent appears beaded on Albert‟s staining?C. Enlist two complication of disease caused by this organism.D. What is the primary mechanism in pathogenesis of Toxin mediatedcomplications. 8
  9. 9. A. Name the test, which is shown in thephotograph.B. Why this test is performed?C. Strip marked as “A” contains whatsubstance?D. Identify the „Test‟, „Positive‟ and„Negative‟ control and write theappropriate alphabet in the answer for each. 9
  10. 10. Vibrio cholerae produces yellowish colonieson agar as shown in the photograph.A. Name the agar on which V. cholerae aregrown in the photograph.B. This agar belongs to which class/type ofculture medium?C. Comment on the motility of V. cholerae.D. How would you differentiate V. choleraefrom members of enterobacteriaceae? 10
  11. 11. A. Name the given media.B. Name the most likely causative agent in this patient?C. Name the major constituents of this medium.D. How much time is required for visible growth of thisorganism on this medium?  When grown on LJ medium, M. tuberculosis appears as brown, granule-like colonies.  The media must be incubated for a significant length of time, usually four weeks, due to the slow doubling time of M. tuberculosis compared with other bacteria (15-20 hours). 11
  12. 12. A pus swab received in the laboratory was inoculatedon blood agar, incubated for 24 hours and smear isprepared from the colony of the bacteria and stainedwith Gram stain.Examine the slide under themicroscope andclassify the organism on the basis of Gram staining.Name the reagents required for Gram staining.Write three conditions, which result in gram variability. 12
  13. 13. Sputum is received from a suspected case of pulmonarytuberculosis and stained smear is provided.A. Examine the slide and write your observation.B. Which staining method has been opted for staining ofthis organism?C. Enumerate the reagents used in this staining method.D. Name the mordant used in this staining method.E. Name the organism, which require modified ZN staining. • Requirements: • Response sheet Pen / ball point Table • Chair / Stool Binocular Microscope • Z N stained slide positive for Mycobacterium tuberculosis. 13 Gold standart of T.B….to confirm it Z.N stain
  14. 14. In a remote village of Punjab, most of the population presented withsimilar complains of sudden onset of vomiting and profuse diarrheawith abdominal cramps. On examination, stool is like rice water inappearance and contains mucous. Stool samples are collected in atransport medium provided to you and to be sent to referral laboratoryfor isolation of the causative organism.Identify the transport medium provided.Write the name of enrichment medium for stool samples.Write two characteristic features of transport media.Alkaline Peptone Water is an enrichment medium used for the cultivation ofVibrio species from feces and other infected materials.Peptones provide nitrogen, vitamins, minerals and amino acids essential forgrowth.Sodium chloride supplies essential electrolytes for transport and osmotic 14balance and encourages the growth of Vibrio cholerae.
  15. 15.  Sabouraud agar is used to culture fungi and has a low pH that inhibits the growth of most bacteria; also contains the antibiotic gentamicin to specifically inhibit the growth of Gram-negative bacteria. 15
  16. 16.  Salmonella shigella agar plate with H2S producing colonies Black color colonies 16
  17. 17. Sensitivity plate 17
  18. 18. Urease Test
  19. 19. Anaerobic Culture Methods Anaerobic jar• Anaerobic jar Figure 6.5
  20. 20. Bacterial StainsAlbert stains - reagents used to demonstrate volutin granules and cytoplasm indiphtheria organisms.Fields Stain - a rapid stain used commonly on thin films for malarial parasites.Flagella Stain - allows for the direct observation of flagellaGentian Violet Stain Recipe - Reagent used in the gram-stain procedure.
  21. 21. LIKE MYPRESENTATION?For Comments & Abbas Naseem Dated: Nov 7, 2010