1. PRACTICAL MICROBIOLOGY
Dr. Waleed Khalid

2. Laboratory Safety Rules,
Microscope
and
Preparation of smear

3. Laboratory Safety Rules
There are specific safety rules that are needed to be followed while working in microbiology lab. These safety rules include :
1-Wear a lab coat in the lab.

4. 2-Eating, drinking and smoking are forbidden in microbiology lab.
3-Thoroughly wash your hands with soap and water before and after lab.
4-Clean the lab bench with disinfectant before and after lab.

5. 5-Dispose of all contaminated materials and slides in specific containers.
6-Bacterial loop has to be sterilized by flame before and after use.

6. LIGHT MICROSCOPE
- A basic microscope consists of two lenses. The uppermost lens, called the ocular lens which is the part through which a person looks. The lower lens is the objective lens. Usually, several objective lenses are localized on a turret, allowing rapid changing of objective lenses. The eyepiece tube holds the ocular and objective lenses in place. Most microbiological specimens are mounted on glass slides and placed on the stage.

8. Procedure : 1 - Clean your lenses with lens paper.
2 - Set the microscope on the scanning lens (red lens (4*)).
3 - Focus using the coarse adjustment.

9. 4 - Change to low power lens (yellow lens (10*)) and focus.
5 - Switch to high power lens (blue lens (40*)). Only use the fine adjustment knob.
6 - Switch the objective to half way between the high power and the oil immersion lens (black and white) (100*) .
7 - Place a drop of oil on the slide.
8 - Turn oil immersion lens into the oil.
9 - Check your image and only use fine to adjust.

11. PREPARATION OF SMEAR :-
Preparation of fixed smear :-
A - From fluid material : ( such as broth culture , urine, sputum , pus , purulent exudates …, etc)
1 - Sterilize the loop in benzene flame , and let it to cool.

12. 2 - Using a septic technique , withdraw a loopful of the specimen and spread it on the center of a clean slide to form a somewhat thick film of 1-2 cm in diameter , then re-sterilize the loop.

13. 3 - Allow the film to dry by air without heating.
4 - The film is fixed on the slide by passing it 3 times through the benzene flame, allow the slide to cool before staining.

14. B - From solid material: ( such as a culture on agar i.e colonies ) :
1 - Sterilize the loop in benzene flame and let it to cool.
2 - Place a loopful of a clean water ( tap water can be used ) on the center of a clean slide.
3 - By the resterilized loop, transfer a small portion of the colony to the drop of water, emulsify thoroughly and spread the mixture evenly on the slide to form a thin film of 1-2 cm of diameter.
4 - Dry and fix as mentioned above

15. AIM OF FIXATION: 1 - Kill the microorganism
2 - Make the microorganism stuck to the surface of the slide
3 - Make the microorganism more permeable to the stain
4 - Prevent the microorganism from going autolytic changes.

16. LAB: 2
STAINING METHODS

17. STAINING METHODS: A - Simple staining technique:
- Simple stains are used to demonstrate the presence of organisms and the nature of any cell present in the smear by applying only one dye.
Stains in general can be divided into three groups: Basic , Acidic and Neutral .
Acidic Stains
Basic Stains
Neutral Stains
Nigrosin
Crystal violet
Giemsa
Malachite green
Methylene blue
Leishman
Acid fuchsin
Safranin
Wright
Basic fuchsin

18. As bacterial cells are rich in nucleic acid ( which has a negative charge) it will follow that “basic stain ,bearing its coloring matter in the positive charge , will be attracted to the organism and stain it” . Acid stain ,however, will not stain the bacteria ; they are used mainly for staining the background material a counterstaining color.

19. Procedure of simple staining:
1. Flood the slide with loefflers methylene blue for 5-10min.
2. Wash off the stain with slowly running tap water.
3. Allow the to slide to dry in air or placed it between two sheets of filter paper.
4. Examine under oil immersion.

20. B.Differentional (compound) staining technique:
- It consists of more than one dye used successively to identify organisms according to their type of reaction.
- The most important examples for this group of stains are:
1. Gram's staining method
2. Acid fast stain (zeihl neelsen methods)
3. Spore stain

21. GRAM STAINING METHOD:
- It is one of the most important methods widely used in bacteriology dicovered in 1884 by gram (a Danish physician), using two dyes in sequence each of different color. He found that bacteria fall into two different categories:
A) Those that retained the first dye (crystal violet) throughout the staining procedure are known as “GRAM POSITIVE”

22. B) Those that lost the first dye (crystal violet)after washing with adecolorizing solution and stained with the second dye (safranine) are known as “GRAM NEGATIVE”

23. - IN CONCLUSION, the gram positive bacteria appear violet ,while gram negative bacteria are red in color .therefore ,it is possible to differentiate between bacteria of the same morphology. furthermore, it can be used to determine the relative number and morphology of bacteria in a smear taken directly from a patient

24. PROCEDURE OF GRAM STAINING:
1 - Flood the slide with crystal or gention violet,leave to act for 1-2min. ,wash with tap water.
2 - Apply gram's iodine (a mordant),leave to act for one minute ,wash with tap water.
3 - Apply 95%ethyl alcohol (a decolorizer).leave to act for seconds ,wash with tap water.
4 -Apply saffranin (the counter stain), leave to act for 1-1.5min. , wash with tap water, blot, dry in air and examine with oil immersion lens.

28. MECHANISM OF STAINING:
The division of bacteria into two categories, indicates a basic chemical differences between gram positive and gram negative bacteria. The most important differences are:
1 - The cell wall of Gram-negative organisms have relatively little peptidoglycan and mainly consist of lipoproteins and polysaccharides. While in Gram positive organisms the peptidoglycan comprises the major part of the cell wall rendering them more rigid than Gram negative cells and less permeable for the dye iodine complex to diffuse freely out of the cell during the process of decolourization.

29. 2 - The more acid charater of the protoplasm of Gram positive bacteria which is enhanced by treatment with iodine may partly explain their stronger retention of the basic dye.
3 - Integrity of the cell wall.

30. LAB:3 CULTURE MEDIA

31. CULTURE MEDIA
- Culture media are used for the recognition and identification of microorganism. The media are contained either in test tubes, plates (Petri dishes) flasks or screw capped bottles which must be thoroughly cleaned before use, then the medium and the container are subsequently sterilized by heat.

32. (Fluid culture medium)
nutrient broth
(Fluid culture medium)
Blood agar and nutrient agar
(Solid culture medium)

33. Common ingredients of culture media
1. Peptone 2.Meat extract 3.Nacl 4. Agar agar a. Melting point c b. solidifing point c c. concentration 1.5-2%
Most pathogenic bacteria have restricted pH (7.2 – 7.4 ) . Therefore, pH of the media should be adjusted using 10% NaOH or HCL

34. TYPES OF CULTURE MEDIA :
- Culture media can be classified according to:
A - Physical state ( consistency ) of the media
1.Liquid (Fluid) media e.g. nutrient broth, peptone water, brain heart infusion . They are commonly used for primary cultivation.

35. 2.Solid media e.g. nutrient agar ,blood agar, MacConkey's agar which are commonly used for cultivation of bacteria.
3. Semisolid media used for cultivation of spirochetes and to study motility. They contain 0.4 – 0.8 % of agar agar.

36. (Fluid culture medium)
nutrient broth
(Fluid culture medium)
Blood agar and nutrient agar
(Solid culture medium)

37. B. According to the use of media
1)Simple or basal media e.g. nutrient agar and nutrient broth.
They are used for the cultivation of common microorganism but not for the fastidious bacteria.

38. Nutrient agar Nutrient broth
Simple culture media

39. 2)special-purpose media e. g
2)special-purpose media e.g. enriched, selective, differential, transport, sensitivity test, etc…
ENRICHED MEDIA :-
-Simple media enriched with appropriate substance ,e.g. Blood ,glucose ,serum and ascetic fluid ,most commonly used to cultivate fastidious microorganism like streptococci.

40. Chocolate agar Blood agar (Enriched media)

41. SELECTIVE MEDIA
-Containing inhibitory substance ( e.g. bile salt ,antibiotic, dyes…etc) which favour the growth of concerned microorganism and inhibit the growth of other . e.g. Maconkey's agar, Potassium tellurite agar, Bismuth sulphite agar , Deoxycholate citrate agar…etc

42. Slective medium for gram negative bacilli (contains bile salts which inhibit the growth of gram positive microorganisms)

43. DIFFERENTIAL MEDIA
-Certain species produce characteristic growth that can be easily recognized, or can produce certain effects in the media e.g. hemolytic and non-hemolytic species on blood agar ,MacConkeys agar differintiates between lactose fermenter and non lactose fermenter gram negative bacilli.

44. Haemolytic bacteria on blood agar
Non haemolytic bacteria on blood agar

45. MacConkey′s agar (selective and differential medium) selective for gram negative bacilli and differentiate between lactose fermenter and non lactose fermenter gram negative bacilli)

46. Staphylococcus albus (white colonies) on nutrient agar
Staphylococcus citreus (lemon yellow colonies) on nutrient agar
Staphylococcus aureus (golden yellow colonies) on nutrient agar

47. Antibiotic susceptibility testing on Mueller-Hinton agar
- This test is used to know the appropriate antimicrobial agents to be used for the treatment of the causative bacteria.
Disk diffusion test
-The culture media is inoculated by the pathogenic bacteria and a paper discs impregnated by different type of antibiotic (Each disc for specific antimicrobial agent eg disc for ampicillin and other for gentamicin, amikacin rifampicin …etc) are placed on this culture media.

48. -The culture is incubated at 37 C for hr according to the type of mo. The test results are read as the following:
1- A Zone of inhibition of growth (no growth around the disc) the bacteria is sensitive to this agent.
2- If the growth of mo reached the edge of the disc this mo is resistant to this antimicrobial drug.

49. Growth to the edge of the disc
No zone of no no inhibition
Growth to the edge of the disc
resistant
Zone of inhibition
No growth of m.o
sensitive

51. LAB: 4 Culture of microorganisms from environment

52. - Microorganisms are found throughout the environment
- Microorganisms are found throughout the environment. For better study of these microorganisms pure culture is needed. Pure culture: a single kind of microorganism growing alone in a protected environment. There are two methods used widely for preparation of pure culture from mixed population. 1. streak plate method. 2. pour plate method.

53. Streak plate method ( Quadrant plate)
This method is used for the isolation of pure culture of bacteria from mixed population e.g. sputum, urine, stool or from pus of infected wound or abscess.

54. Technique of Streak plate method :
Sterilize the loop in a benzene flame, cool it and streak the specimen over an area-A.
Re-sterilize the loop, cool it, then streak over an area-B from the distal part of area-A.
Continue the streak in the same manner for areas C and D.
Incubate the plates at 37°C for 24 hours.

56. A subculture is done after 24 hours incubation from one of the colonies needed for study on a sterile medium following the same technique mentioned previously. The culture produced is a pure one.
coli                                     

57. Pour plate method
-This method involved serial dilution of specimen and mixed with melted media. Then pour the content into sterile plate plates.

58. Cultural characteristics:
Cultural characteristics involve both :
1. Growth requirement.
2. Colonial morphology.
It is used to study the macroscopic characteristics of a pure culture on a solid medium.

59. Colonial morphology
The following points have to be considered in describing a colony:
1. Size: measured by mm.

60. 2. Shape: rhizoid ,circular, filamentous, irregular.

62. 3. Elevation: flat, convex, raised, umbonate.

63. 4. margin: entire lobulated ,filamentous

64. 5. Consistency: dry, mucoid.

65. 6. Surface texture: rough, smooth.

66. 7. Color or pigmentation.

68. 8. Optical density : opaque, translucent, glistening.

69. 9. changes in the inoculated medium e.g heamolysis.
10. Odour : bad or sweat musty odour.

70. Stock culture :-
Means preservation of a microorgansim in a culture medium for future study. It can be kept usually for 1 month at 4 °C.

71. Technique of stock culture:
Use a universal bottle containing medium in slanted position ( to provide a wide surface for inoculation and good nutrition).
Using a sterile loop inoculate the surface of the medium with one or few colonies of a pure culture.
Incubate the inoculated medium at 37 °C for 24 hours, then store at 4 °C.