1. Methods in Clinical and diagnostic Microbiology (Part Two)
Dr. Majid Zare Bidaki
Associate Professor
Birjand University of Medical Sciences

2. Microscopic observation

3. General types of preparation microscopy in microbiology
Wet smear microscopy
Hanging drop

4. Microscopic methods Wet smear with no staining with staining
Dried smear
Positive staining
Simple staining
Complex staining
Negative staining

5. Dried smear microscopy
Smear preparation
Drying
Fixation
Staining
Washing

6. Negative staining

7. Negative staining

8. Positive staining

9. Positive staining (see the clip !)
Simple staining
Only one colour
Complex staining (Differential staining)
Two colour
Decolorization componant
Colour fixator

10. Gram staining
History
Importance and applications
Stages
Mechanism

13. Other differential staining
Acid fast
Albert
Spore
Flagella
Capsule

15. Acid fast

16. Spore staining

18. Albert staining

21. For Microscopic observation: Staining components

22. Microscopy equipments and tools

23. Bacterial Culture

24. Equipment for culture: Incubator

25. Refrigerator Equipment for keeping components, reagents and prepared madia

26. Refrigerator

27. For Microbial culture: Different types of Media

28. Agar
Composed of two long-chain polysaccharides (70% agarose and 30% agarapectin).
melts at 95oC and solidifies at 42oC.
it is not hydrolyzed by most bacteria and is usually free from growth promoting or growth retarding substances.
commonly, used at concentration of 1-3% to make a solid agar medium. Reducing the amount of agar to % for semi-solid media.
Stuart’s, Amies media, Hugh & Leifson’s Oxidation Fermentation (OF) as well as SIM motility medium are semi-solid.

29. Medium Types based on solidity: 1. Liquid medium BHI, TSB, SF, NB, …
2. Solid medium
Blood agar, Nutrient agar, chocolate agar, Columbia agar, EMB
3. Semi-solid medium
SIM
4. Biphasic media
Castaneda system

34. Undefined medium & Defined medium
A Undefined medium is a basal or complex medium.
Contains:
- A source of amino acids and nitrogen (e.g., beef extract, yeast extract)
- A carbon source. such as glucose
- Water
- Various salts

35. Defined medium
A defined medium (a synthetic medium) is a medium in which
- all the chemicals used are known
- no yeast, animal or plant tissue is present

36. Nutrient agar medium composition:
Is this an Undefined medium or Defined medium ? Nutrient media (Broth/Agar)
Nutrient agar medium composition:
- Beef Extract-0.3gm (mineral and carbohydrate)
- Peptone-0.5gm (protein and nitrogen source)
- NaCl-0.5gm (electrolyte)
- Agar-1.5gm (solidifying agent)
- Distilled water-100ml pH-7

37. Minimal media
contain the minimum nutrients possible for colony growth, generally without the presence of amino acids.
used to grow "wild type" microorganisms and select for or against recombinants.
Minimal medium typically contains:
A carbon source (such as glucose, or a less energy-rich source like succinate.
- various salts (provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria to synthesize protein and nucleic acid
- Water

38. Supplementary minimal media
A type of minimal media that also contains a single selected agent, usually an amino acid or a sugar.
This supplementation allows for the culturing of specific lines of auxotrophic recombinants.
Auxotrophic: An organism unable to synthesize a particular organic compound required for its growth. 

39. Culture media based on the application in clinical labs
General medium (Nutrient agar, Muller hinton agar, Nutrient broth, …)
Selective/Special medium (EMB, MSA, Palcam Agar Base …)
Differential medium (MAC [MacConkey agar], EMB, XLD, …)
Enrichment medium (SF, RV [Rappaport-Vassiliadis Broth], TT, alkaline peptone water, Listeria Enrichment Medium, …)

40. Culture media (cont.)
Enriched medium (Blood agar, Chocolate agar, cold enrichment)
Transport medium (Stuart, Cary-Blair, Amies, …)
Cary-Blair: exclusively associated with enteric transport
Stuart & Amies: widely used for the transportation of a diverse range of clinical swab samples from sites including the eye, ear, nose, throat, skin, genital tract and wounds.
Galleries (TSI, SIM, MRVP, Citrate agar, ….)

41. Transport media

42. Main methods of bacterial culture
Culture for isolation
for example: Streak plate method
Culture for counting
for example for urine culture
Spread plate culture
For example for antibiogram

43. Streak plate method

44. Colony count culture

45. Spread plate culture

46. Spread plate culture

47. Serologic methods Antigen - Antibody reactions
Antibodies (Antiserums) & Antigens

48. ELISA: to detect serologic reaction

49. ELISA: to detect serologic reaction

50. Genetic methods
Polymerase chain reaction (PCR)

51. For Polymerase chain reaction (PCR)
DNA extraction: Filter and lysozyme
PCR: Taq polymerase, dNTPs, Primers, template, buffers

52. Genetic methods
Termocycler

53. PCR
Gel electrophoresis tank
Termocycler
Gel duck