MICROBIOLOGY PRACTICAL

INVADERS
Unit

:I

Problem

:Invaders

Year

:2

Date

:Tuesday 09.10.2012

Time

:09:00-11:00 a.m...
STATION-1:
1.Observe and discuss the different types of
culture media
ROUTINE LABORATORY MEDIA
1. Basal media, e.g. Nutrie...
TYPES OF CULTURE MEDIA
Media are of different types on consistency and chemical
composition.
A. On Consistency:

1. Solid ...
B. On Chemical Composition :
1. Routine Laboratory Media
2. Synthetic Media. These are chemically
defined media prepared f...
1. BASAL MEDIA.
Basal media are those that may be used for growth (culture)
of bacteria that do not need enrichment of the...
4. INDICATOR (DIFFERENTIAL) MEDIA.
An indicator is included in the medium. A particular
organism causes change in the indi...
TYPES OF CULTURE MEDIA
BASAL MEDIA
Basal media are those that may be used for growth (culture)
of bacteria that do not nee...
TYPES OF CULTURE MEDIA
Enrichment media
Enrichment media: This is a media which promotes
the growth of a particular organi...
TYPES OF CULTURE MEDIA
Differential Medium:
Culture medium that allows one to distinguish between
or among different micro...
TYPES OF CULTURE MEDIA
Selective Medium:
Culture medium that allows the growth of
certain types of organisms, while inhibi...
TRANSPORT MEDIA
These media are used when specie-men cannot be
cultured soon after collection. Examples: Cary-Blair
medium...
NUTRIENT AGAR
A general purpose medium which may be enriched with 10% blood or other biological fluid.
Formula
'Lab-Lemco'...
Blood Agar
Tryptone

15 g

Phytone or soytone

5g

NaCl
Agar
Distilled water

5g
15 g
1 liter

Heat with agitation to diss...
MacConkey Agar
Proteose peptone or polypeptone

3g

Peptone or gelysate

17 g

Lactose

10 g

Bile salts No. 3 or bile sal...
SABOURAUD DEXTROSE AGAR
Sabouraud Dextrose Agar is used for the cultivation of fungi.
Product Summary and Explanation
Sabo...
Continue SABOURAUD DEXTROSE AGAR
Expected Cultural Response
Cultural response on Sabouraud Dextrose Agar at 25 - 30°C afte...
Continue SABOURAUD DEXTROSE AGAR
Expected Cultural Response
Cultural response on Sabouraud Dextrose Agar at 25 - 30°C afte...
Anaerobic Transport Media Surgery Pack (ATMSP)
Anaerobic Transport Medium Surgery Pack (ATMSP) is a mineral salt base semi...
Edited by: A. Qareeballa
STATION-1:
2. Observe and discuss the atmospheric
conditions (O2 requirement, incubation
temperature and pH) for microorga...
Ordinary Incubator
Most of the medically important organisms require
incubation temperature of: 35oC - 37oC
Edited by: A. ...
Co2 Incubator
Anaerobic Incubator for anaerobes

Edited by: A. Qareeballa
Anaerobic Jar

Microaerophilic Jar
Edited by: A. Qareeballa
Candle Jar for CO2
Edited by: A. Qareeballa
Edited by: A. Qareeballa
Anaerobic chamber

Edited by: A. Qareeballa
STATION-1
3. Examine macroscopically:
• Culture plates showing bacterial colonies
• Antimicrobial susceptibilty testing pl...
NUTRIENT AGAR
Cultured with some
organisms
Notice: Bacterial
colonies

Edited by: A. Qareeballa
Hemolysis with Blood Agar

Edited by: A. Qareeballa
Edited by: A. Qareeballa
SABOURAUD DEXTROSE AGAR
Cultured with Candida species

Edited by: A. Qareeballa
Molds growth on Sabouraud agar
Edited by: A. Qareeballa
STATION-2
1.Explain the preparation of bacteriological
smears and stain the provided prepared smear
with Gram’s staining m...
Gram staining technique
Required:
Basic Stain
Mordant
 Crystal violet  Lugol's
 or Gentian
iodine
violet

Decolorizer:
...
PREPARATION OF THE SMEAR
1. A small sample of a bacterial culture is removed from
a culture. In this example it is being t...
IMPORTANT NOTES:
• Failure to follow these directions may cause
staining artifacts and disrupt the normal
morphology of ba...
Gram staining technique
Method
1. Fix the dried smear using gentle heat or fix with methanol for 2 minutes. The purpose of...
STATION-2
2. Examine microscopically Stained smears
of::
• Gram-positive cocci.
• Gram-negative cocci
• Gram-negative baci...
Edited by: A. Qareeballa
Amastigote of Leishmania sp.

Promastigote of Leishmania sp.

Edited by: A. Qareeballa
VERO Cells, normal

Infected with herpes B Virus
Phase-contrast microscopic image
of Vero cells.
Edited by: A. Qareeballa
Invaders 09 10-2012
Upcoming SlideShare
Loading in...5
×

Invaders 09 10-2012

659

Published on

Published in: Education, Technology, Business
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
659
On Slideshare
0
From Embeds
0
Number of Embeds
0
Actions
Shares
0
Downloads
31
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Invaders 09 10-2012

  1. 1. MICROBIOLOGY PRACTICAL INVADERS Unit :I Problem :Invaders Year :2 Date :Tuesday 09.10.2012 Time :09:00-11:00 a.m OBJECTIVES: Each student should be familiar with the basic techniques for the identification of medically important microorganisms (Bacteria, Fungi, Parasites and Viruses): MATERIALS MAY BE POTENTIALLY INFECTIOUS, YOU ARE EXPECTED TO ADOPT THE UNIVERSAL PRECAUTIONS (UP) Edited by: A. Qareeballa
  2. 2. STATION-1: 1.Observe and discuss the different types of culture media ROUTINE LABORATORY MEDIA 1. Basal media, e.g. Nutrient agar. 2. Enriched media, e.g. blood agar. 3. Enrichment media, e.g. Selenite F 4. Differential (Indicator media), e.g. MacConkey agar. 5. Selective media, e.g. Campylobacter medium. 6. Transport media 7. Storage media. 8. Leishmania media 9. Anaerobic media, e.g. (Cooked meat & Thioglycholate media) Edited by: A. Qareeballa
  3. 3. TYPES OF CULTURE MEDIA Media are of different types on consistency and chemical composition. A. On Consistency: 1. Solid Media. a. Advantages of solid media: • Bacteria may be identified by studying the colony character, • Mixed bacteria can be separated. b. Solid media is used for the isolation of bacteria as pure culture. 'Agar' is most commonly used to prepare solid media. • Agar is polysaccharide extract obtained from seaweed. • Agar is an ideal solidifying agent as it is : • Bacteriologically inert, i.e. no influence on bacterial growth, • It remains solid at 37°C, and • It is transparent. 2. Liquid Media. It is used for profuse growth, e.g. blood culture in liquid media. Mixed organisms cannot be separated. Edited by: A. Qareeballa
  4. 4. B. On Chemical Composition : 1. Routine Laboratory Media 2. Synthetic Media. These are chemically defined media prepared from pure chemical substances. It is used in research work Edited by: A. Qareeballa
  5. 5. 1. BASAL MEDIA. Basal media are those that may be used for growth (culture) of bacteria that do not need enrichment of the media. Examples: Nutrient broth, nutrient agar and peptone water. Staphylococcus and Enterobacteriaceae grow in these media. 1. ENRICHED MEDIA. The media are enriched usually by adding blood, serum or egg. Examples: Enriched media are blood agar and Lowenstein-Jensen media. Streptococci grow in blood agar media. 2. SELECTIVE MEDIA. These media favour the growth of a particular bacterium by inhibiting the growth of undesired bacteria and allowing growth of desirable bacteria. Examples: MacConkey agar, Lowenstein-Jensen media, tellurite media (Tellurite inhibits the growth of most of the throat organisms except diphtheria bacilli). Antibiotic may be added to a medium for inhibition. Edited by: A. Qareeballa
  6. 6. 4. INDICATOR (DIFFERENTIAL) MEDIA. An indicator is included in the medium. A particular organism causes change in the indicator, e.g. blood, neutral red, tellurite. Examples: Blood agar and MacConkey agar are indicator media. 5. TRANSPORT MEDIA. These media are used when specie-men cannot be cultured soon after collection. Examples: Cary-Blair medium, Amies medium, Stuart medium. 6. STORAGE MEDIA. Media used for storing the bacteria for a long period of time. Examples: Egg saline medium, chalk cooked meat broth. Edited by: A. Qareeballa
  7. 7. TYPES OF CULTURE MEDIA BASAL MEDIA Basal media are those that may be used for growth (culture) of bacteria that do not need enrichment of the media. Examples: Nutrient broth, nutrient agar and peptone water. Staphylococcus and Enterobacteriaceae grow in these media. ENRICHED MEDIA Enriched media: Blood and other special nutrients may be added to general purpose media to encourage the growth of fastidious microbes. These specially prepared media are called as enriched media. e.g. Blood agar, Chocolate agar. Edited by: A. Qareeballa
  8. 8. TYPES OF CULTURE MEDIA Enrichment media Enrichment media: This is a media which promotes the growth of a particular organism by providing it with the essential nutrients and rarely contains certain inhibitory substance to prevent the growth of normal competitors. e.g. Selenite F broth- this media favours the growth of Salmonella also prevents the growth of normal competitors like E. coli . but E. coli do not perish in the medium but they do not flourish like Salmonella Edited by: A. Qareeballa
  9. 9. TYPES OF CULTURE MEDIA Differential Medium: Culture medium that allows one to distinguish between or among different microorganisms based on a difference in colony appearance (color, shape, or growth pattern) on the medium. Dyes in the medium (e.g.: eosin/methylene blue in EMB) or pH indicators change the color of the medium as sugars in the medium (e.g.: lactose in EMB & MacConkey's and Mannitol in MSA) are fermented to produce acid products Edited by: A. Qareeballa
  10. 10. TYPES OF CULTURE MEDIA Selective Medium: Culture medium that allows the growth of certain types of organisms, while inhibiting the growth of other organisms Dyes in the medium (e.g.: methylene blue in EMB & crystal violet in MacConkey medium) or high salt concentration in the medium (e.g.: 7% salt in Mannitol Salt Agar) inhibit the growth of unwanted microorganisms Edited by: A. Qareeballa
  11. 11. TRANSPORT MEDIA These media are used when specie-men cannot be cultured soon after collection. Examples: Cary-Blair medium, Amies medium, Stuart medium. STORAGE MEDIA Media used for storing the bacteria for a long period of time. Examples: Egg saline medium, chalk cooked meat broth. Edited by: A. Qareeballa
  12. 12. NUTRIENT AGAR A general purpose medium which may be enriched with 10% blood or other biological fluid. Formula 'Lab-Lemco' powder Yeast extract Peptone Sodium chloride Agar pH gm/litre 1.0 2.0 5.0 5.0 15.0 7.4 +/- 0.2 Directions Suspend 28g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilize by autoclaving at 121°C for 15 minutes. Description Nutrient Agar is a basic culture medium used to subculture organisms for maintenance purposes or to check the purity of subcultures from isolation plates prior to biochemical or serological tests. In semi-solid form, agar slopes or agar butts the medium is used to maintain control organisms. Nutrient Agar is suitable for teaching and demonstration purposes. It contains a concentration of 1.5% of agar to permit the addition of up to 10% of blood or other biological fluid, as required. The medium, without additions may be used for the cultivation of organisms which are not exacting in their food requirements. For a medium which is richer in nutrients, see Blood Agar Base. Edited by: A. Qareeballa
  13. 13. Blood Agar Tryptone 15 g Phytone or soytone 5g NaCl Agar Distilled water 5g 15 g 1 liter Heat with agitation to dissolve agar. Autoclave 15 min at 121°C. Cool to 50°C. Add 5 ml defibrinated sheep red blood cells to 100 ml melted agar. Mix and pour 20 ml portions into sterile petri dishes. Final pH of base, 7.3 ± 0.2. Edited by: A. Qareeballa
  14. 14. MacConkey Agar Proteose peptone or polypeptone 3g Peptone or gelysate 17 g Lactose 10 g Bile salts No. 3 or bile salts mixture 1.5 g NaCl 5g Neutral red 0.03 g Crystal violet 0.001 g Agar 13.5 g Distilled water 1 liter Suspend ingredients and heat with agitation to dissolve. Boil 1-2 min. Autoclave 15 min at 121°C, cool to 45-50°C, and pour 20 ml portions into sterile petri dishes. Dry at room temperature with lids closed. Final pH, 7.1 ± 0.2. Edited by: A. Qareeballa
  15. 15. SABOURAUD DEXTROSE AGAR Sabouraud Dextrose Agar is used for the cultivation of fungi. Product Summary and Explanation Sabouraud Dextrose Agar (SDA) is used for cultivating pathogenic & commensal fungi and yeasts. The high dextrose concentration and acidic pH of the formula permits selectivity of fungi. SDA enhanced with the addition of cycloheximide, streptomycin, and penicillin to produce an excellent medium for the primary isolation of dermatophytes. Sabouraud Dextrose Agar is used clinically to aid in the diagnosis of yeast and fungal infections. Principles of the Procedure Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue provide the nitrogen and vitamin source required for organism growth in Sabouraud Dextrose Agar. The high concentration of Dextrose is included as an energy source. Agar is the solidifying agent. Formula / Liter Enzymatic Digest of Casein......................................................5 g Enzymatic Digest of Animal Tissue...........................................5 g Dextrose..................................................................................40 g Agar.........................................................................................15 g Final pH: 5.6 ± 0.2 at 25°C Formula may be adjusted and/or supplemented as required to meet performance specifications. Directions 1.Suspend 65 g of the medium in one liter of purified water. 2.Heat with frequent agitation and boil for one minute to completely dissolve the medium. 3.Autoclave at 121°C for 15 minutes. Edited by: A. Qareeballa
  16. 16. Continue SABOURAUD DEXTROSE AGAR Expected Cultural Response Cultural response on Sabouraud Dextrose Agar at 25 - 30°C after 2 – 7 days of incubation. Microorganism Response Aspergillus niger ATCC® 16404 growth Candida albicans ATCC® 10231 growth Microsporum canis ATCC® 36299 growth Penicillium roquefortii ATCC® 10110 growth Trichophyton mentagrophytes ATCC® 9533 growth Results Yeasts grow creamy to white colonies. Molds will grow as filamentous colonies of various colors. Count the number of colonies and consider the dilution factor (if the test sample was diluted) in determining the yeast and/or mold counts per gram or milliliter of material. Edited by: A. Qareeballa
  17. 17. Continue SABOURAUD DEXTROSE AGAR Expected Cultural Response Cultural response on Sabouraud Dextrose Agar at 25 - 30°C after 2 – 7 days of incubation. Microorganism Response Aspergillus niger ATCC® 16404 growth Candida albicans ATCC® 10231 Growth Microsporum canis ATCC® 36299 Growth Penicillium roquefortii ATCC® 10110 growth Trichophyton mentagrophytes ATCC® 9533 growth Results Yeasts grow creamy to white colonies. Molds will grow as filamentous colonies of various colors. Count the number of colonies and consider the dilution factor (if the test sample was diluted) in determining the yeast and/or mold counts per gram or milliliter of material. Edited by: A. Qareeballa
  18. 18. Anaerobic Transport Media Surgery Pack (ATMSP) Anaerobic Transport Medium Surgery Pack (ATMSP) is a mineral salt base semi-solid media with reducing agents designed as a holding medium for maintaining viability of microorganisms, especially anaerobic bacteria through collection, transport and shipment of clinical specimens from a sterile surgical environment. The Anaerobic Transport Medium Surgery Pack contains buffered mineral salts in a semi-solid media with sodium thioglycolate and cysteine added to provide a reduced environment. Resazurin is added as a redox indicator to reveal exposure to oxygen by turning blue. This product has been prepared to provide an environment, which maintains viability of most microorganisms without significant multiplication and allows for dilution of inhibitors present in clinical material. This medium is designed to meet the stringent viability requirements of obligate anaerobes. All items are supplied with screw caps containing rubber septa (Hungate caps), which allows for either direct injection of aspirated clinical material or introduction of tissue samples. The contents and outer surface of the tube are sterile. This medium is prepared, dispensed and packaged under oxygen-free conditions to prevent the formation of oxidized products prior to use. Edited by: A. Qareeballa
  19. 19. Edited by: A. Qareeballa
  20. 20. STATION-1: 2. Observe and discuss the atmospheric conditions (O2 requirement, incubation temperature and pH) for microorganisms growth: • Aerobic • Anaerobic • Microaerophilic • CO2 requirements. Edited by: A. Qareeballa
  21. 21. Ordinary Incubator Most of the medically important organisms require incubation temperature of: 35oC - 37oC Edited by: A. Qareeballa
  22. 22. Co2 Incubator Anaerobic Incubator for anaerobes Edited by: A. Qareeballa
  23. 23. Anaerobic Jar Microaerophilic Jar Edited by: A. Qareeballa
  24. 24. Candle Jar for CO2 Edited by: A. Qareeballa
  25. 25. Edited by: A. Qareeballa
  26. 26. Anaerobic chamber Edited by: A. Qareeballa
  27. 27. STATION-1 3. Examine macroscopically: • Culture plates showing bacterial colonies • Antimicrobial susceptibilty testing plates • Fungi (molds) contaminated bread. • Cultured plates seeded with Candida species • Adult parasite worms, e.g. Ascaris lumbricoides & Fasciola hepatica Edited by: A. Qareeballa
  28. 28. NUTRIENT AGAR Cultured with some organisms Notice: Bacterial colonies Edited by: A. Qareeballa
  29. 29. Hemolysis with Blood Agar Edited by: A. Qareeballa
  30. 30. Edited by: A. Qareeballa
  31. 31. SABOURAUD DEXTROSE AGAR Cultured with Candida species Edited by: A. Qareeballa
  32. 32. Molds growth on Sabouraud agar Edited by: A. Qareeballa
  33. 33. STATION-2 1.Explain the preparation of bacteriological smears and stain the provided prepared smear with Gram’s staining method. 2.Examine microscopically: • Gram-positive cocci. • Gram-negative cocci • Gram-positive bacilli (rods) • Gram-negative bacilli (rods) • Yeast cells. • Protozoa, e.g. Leishmania species. Edited by: A. Qareeballa
  34. 34. Gram staining technique Required: Basic Stain Mordant  Crystal violet  Lugol's  or Gentian iodine violet Decolorizer:  Acetone  or Acetonealcohol  or Ethanol Counterstain:  Neutral red (1% w/v)  or Safranin  or Diluted carbol fuchsin Edited by: A. Qareeballa
  35. 35. PREPARATION OF THE SMEAR 1. A small sample of a bacterial culture is removed from a culture. In this example it is being taken from a broth culture of the pure microbe but it could be removed from a culture on solid medium or from material containing bacteria eg faeces or soil. 2.The bacterial suspension is smeared onto a clean glass slide. If the bacteria have been removed from a culture on solid media or it is from a soil or faeces sample it will have to be mixed with a drop of bacteria-free saline solution. 3.The bacterial smear is then dried slowly at first and then, when dry, heated for a few seconds to the point when the glass slide is too hot to handle. This fixes ie kills the bacteria making the slide safe to handle. Care must be taken not to overheat which will char the cells Edited by: A. Qareeballa
  36. 36. IMPORTANT NOTES: • Failure to follow these directions may cause staining artifacts and disrupt the normal morphology of bacteria and cells. • To be visible on a slide, organisms that stain by the Gram method must be present in concentrations of a minimum of 104 to 105 organisms/ml of unconcentrated staining fluid. At lower concentrations, the Gram stain of a clinical specimen seldom reveals organisms even if the culture is positive. • Smears that are not properly fixed tend to be washed away during staining and washing resulting in the absence of stained bacteria. Edited by: A. Qareeballa
  37. 37. Gram staining technique Method 1. Fix the dried smear using gentle heat or fix with methanol for 2 minutes. The purpose of fixation is to preserve microorganisms and prevent smears being washed from slides during staining. 2. Cover the fixed smear with crystal violet stain for 30-60 seconds. 3. Rapidly wash off the stain with clean water. 4. Tip off all the water, and cover the smear with Lugol's iodine for 30-60 seconds. 5. Wash off the iodine with clean water. 6. Decolorize rapidly {few seconds} with acetone-alcohol. Wash immediately with clean water. Caution: Acetone- alcohol is highly flammable; therefore use it well away from an open flame. 7. Cover the smear with neutral red stain for 1 minute. 8. Wash off the stain with clean water. 9. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry. 10. Examine the smear microscopically, first with the 40x objective to check the staining and to see the distribution of material, and then with the oil immersion objective (X100) to report the bacteria and cells. Results Gram positive bacteria Dark purple Yeast cells Dark purple Gram negative bacteria Pale to dark red Nuclei of pus cells Red Epithelial cells Pale red Edited by: A. Qareeballa
  38. 38. STATION-2 2. Examine microscopically Stained smears of:: • Gram-positive cocci. • Gram-negative cocci • Gram-negative bacilli (rods) • Gram-positive bacilli (rods) • Yeast cells. • Protozoa, e.g. Leishmania species. Edited by: A. Qareeballa
  39. 39. Edited by: A. Qareeballa
  40. 40. Amastigote of Leishmania sp. Promastigote of Leishmania sp. Edited by: A. Qareeballa
  41. 41. VERO Cells, normal Infected with herpes B Virus
  42. 42. Phase-contrast microscopic image of Vero cells. Edited by: A. Qareeballa
  1. A particular slide catching your eye?

    Clipping is a handy way to collect important slides you want to go back to later.

×