1. Lab 1

2. Media and media preparation

3. Important equipment in the lab
Microscope
Incubator : bacteria usually at 37c , fungi from c
Autoclave
Safety Cabinet

4. Media in Microbiology
Culture media contains the nutrients needed to allow microbe to grow.
Types of media
- General
- Selective
- Differential
- Selective & differential
Forms of media
- broth
- solid
- semi-solid

5. How is media made?
When lab personnel make media they measure out a quantity of dry powdered nutrient media, then add water
They dispense the media into bottles (flask,tube), cap it and autoclave. The autoclave exposes the media to high temperature (121°C) and pressure (15 psi) for 20 minutes.
Once the media is autoclaved it is sterile
(all microoranism forms killed)

6. How is media made?
1- Suspend x g agar powder in x liter of distilled water.
- measure required amount by using balance
- Then add the powder to a flask
- Add the right amount of d. water
2- Bring to the boil to dissolve completely
3- autoclaving
4- pouring

7. Safety To disinfect bench surfaces , we use Alcohol 70%
Don’t eat or drink in lab
Always wear gloves
Clean bench after use
Wash hands before leaving the lab

8. Lab 2

10. Condenser Lens: The function of the condenser lens is to collect the light from the illuminator and focus it on the specimen.
Diaphragm or Iris: The diaphragm is used to control the amount of light reaching the specimen. In a student scope it is a rotating disk under the stage and above the condenser.
Rules for Using the Microscope
1. Use two hands to carry a microscope, one hand holding the arm, the other
holding the base.
2. Only use lens paper and water to clean the lenses.

11. Magnification : the ability to make small objects seem larger
microscope has 4 magnifications: Scanning, Low , High & oil immersion. Each objective will have written the magnification. In addition to this, the ocular lens (eyepiece) has a magnification. The total magnification is the ocular x objective.
Resolution: the ability to see fine details.
Contrast: the ratio between the dark and the light. (difference in color)
Magnification
Ocular lens
Total Magnification
Scanning 4x
10x
40x
Low Power
100x
High Power
400x
Oil immersion
1000x

12. Numerical Aperture NA
NA means The light-gathering ability of a microscope objective.
The higher the NA value the better the resolution.
For 100x lens , we usually add oil between the lens and slide. why ?
- This is because that the refractive index of air is 1 and oil is ~ 1.5.
So, NA value increases with oil.

13. Lab 3

14. Staining
Simple staining : only one dye . Ex. Simple stain methylene blue
Differential staining: more than one dye . Differentiation among bacteria is possible Ex. Gram stain
Special staining: stain Special structures Ex. Capsule stain , spore stain.

15. Gram stain Hans Christian Gram , Danish scientist
Classify Bacteria into Gram negative & Gram positive .
Thin cell wall
Thick cell wall

16. 4 dyes 1- primary stain: ( crystal violet ) 1 minute
2- mordant : ( iodine ) 1 minute
3- decolorizer : ( alcohol or acetone ) 1-2 seconds
4- counterstain : ( safranin ) 1 minute

17. Lab 4

18. Parasites
multicellular
unicellular

19. Protozoa examples Trypanosoma spp. - flagellated protozoa
- causes sleeping sickness disease

20. Protozoa examples
Plasmodium falciparum
- causes Malaria
- blood smear

21. Helminth: nematodes (roundworm)
Enterobius vermicularis
worm
egg

22. Helminth: nematodes (roundworm)
Ascaris lumbricoides
egg

23. Helminths: trematodes ( Fluke)
Schistosoma
Terminal spine
lateral spine
rudimentary lateral spine

24. Helminth: cestode (tapeworm)
Taenia saginata (beef tapeworm)
worm
egg

25. Fungi
Unicellular or multicellular eukaryotes.

26. Yeast examples
Candida albicans
Pseudohypae, hyphae and yeast

27. Mold examples
Aspergillus spp.
- septated hyphae

28. Rhizopus spp.
- aseptataed hyphae
sporangium
spores

29. Lab 5 &6

30. Important terms colony
- is a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike.

31. Culture vs. subculture
Culture: is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions
Sub-culture: a new cell or microbiological culture made by transferring some or all cells from a previous culture to fresh growth medium.

32. Mixed vs. pure culture
Mixed culture: two or more microgranism on the same plate.

33. Pure culture : only one microorganism on the plate

34. colony morphology Form : circular , filamentous Pigmentation : colors
Texture : dry , moist , mucoid
Surface : smooth, rough, shiny
Size : tiny , large

35. Examples of colonies
Zone of hemolysis on blood agar

36. Examples of colonies
Swarming on plate ex. Proteus spp.

37. Examples of colonies
Mucoid colony ex. Klebsiella spp.

38. Examples of colonies
Irregular colony form ex. Bacillus

39. Streaking
purpose :To have isolated colonies

40. Lab 7

41. The Vegetative Cell Gives Rise to One Spore
Bacterial Cell
Bacterial Cell
Spore
Bacterial Cell
The endospore is able to survive for long periods
of time until environmental conditions again become favorable for growth.
The endospore then germinates, producing a single vegetative bacterium.

42. Not all bacterial species can form spores
A few genera of bacteria produce Endospore such as Clostridium and Bacillus
(Endospore production is associated with Gram Positive bacteria)

44. Schaeffer-Fulton Stain Procedure
1. Prepare a smear. Air Dry. Heat fix
2. Put the slide on steam rack
3. Flood the smear with Malachite Green stain
4. Steam slide for 5 minutes (be sure that the satin is not dry, if so , add more satin )
5. Drain slide and rinse with DI water.
7. Flood smear with Safranin (counter stain). This stains the vegetative cell. (Leave for 1 minute)
8. Drain the slide and rinse with DI water
9. Dry
10. View slide under 100x oil immersion.

45. Endospore Stain Example Spores: Green Cell: Red or Pink
Practical: Each student will make smear of Bacillus subtilis

46. Lab 8

47. Bacterial count

48. Bacterial count
Total count is a count of cells including dead and live cells.
Viable count is a count of only those cells that are alive in the sample.
Also know as “viable cell counts”
Concentrated samples are diluted by serial dilution
The diluted samples can be either plated by spread plating or by pour plating

50. colony-forming unit CFU
- a colony-forming unit is a unit used to estimate the number of viable bacteria cells in a sample

51. Procedure:
- aseptically dilute sample
- make 1:10 dilutions as instructed, based on sample
- aseptically transfer 100 ul volume of dilutions that are to be
plated onto agar plates
- Spread the sample on the agar very well
- incubate as instructed for growth of colonies
- during next lab period, count colonies on plates
- use the formula:
cfu/ml = # of colonies x dilution
amount plated ( ml)

52. Calculation
Example
Bacterial sample is diluted 1:10 (i.e. 1 ml diluted with 9 ml saline)
0.01 ml of 1/10 dilution is inoculated onto AC plates
87 colonies grow
The Concentration of bacteria is
cfu/ml = # of colonies x dilution
amount plated ( ml)
87 x 10
.01
= 87,000 cfu/ml