1. Cellular makeup Prokaryote Eukaryote Figure: 02-01a-b Caption:
Internal structure of microbial cells. (a) Diagram of a prokaryote. (b) Diagram of a eukaryote.
Eukaryote

3. Table 1.2 Subgroups of Microorganisms

4. Perbedaan sel prokariot dan eukariot
Ciri-Ciri
Prokariot
Eukariot
Ukuran sel :
Diameter
0,2-5 m
2-100 m
Struktur genetik :
Membran inti
Nukleolus
Kromosom -
DNA sirkular +
DNA linier
Struktur sitoplasma:
Retikulum endoplasma
Mitokondria
Lisosom
Ribosom
Stioskeleton
Dinding sel mengandung peptidoglikan
70 S
80 S

5. Struktur organel sel Eukaryote (Starr, 1998)

17. How do you study something that you cant see?
You look at it under the microscope
But certain microbes (e.g. bacteria) do not have too many identifying attributes
You grow large populations of them (i.e. culture them).
You observe the behavior of the population under various environmental conditions
On Solid media
In the presence of certain biochemicals
Based on the biochemical reactions they cause

18. compound light microscope

19. Pathway of light

20. Effect of wavelength on resolution

21. Effect of magnification

22. Oil immersion lens

23. magnification – ability to enlarge objects
resolving power – ability to show detail

24. Specimen preparation
wet mounts & hanging drop mounts – allow examination of characteristics of live cells: motility, shape, & arrangement
fixed mounts are made by drying & heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts.

25. Bacterial shapes and arrangements

26. Figure 3.8 Preparing a hanging drop mount.
Wet mounts (to observe living microbes)
- Simple wet mounts : dry out quickly
- hanging drop mounts : Using petroleum jelly to prevent drying of microbe samples

27. Stains
- dyes that increase contrast by binding selectively to certain cells or to certain parts of them
- vital stains : stain living cells, but more effective only after microbes have been fixed
- Fixation : often distorts the cell’s appearance, motility can no longer be studied
Heat fixation – using the open flame
Chemical fixation – does less damage than heat
ex) Osmic acid, formaldehyde,
glutaraldehyde…..

28. Types of dyes
- Basic dyes : with positive charges, stains the surfaces of most microbes
- Acidic dyes : with negative charges, stains negatively charged parts of cells, including proteins
ex) stain animal tissues that microbes have invaded
- Mordants : intensify staining by increasing a cell’s affinity for a dye

29. Staining
cationic dyes - basic, with positive charges on the chromophore
anionic dyes - acidic, with negative charges on the chromophore
surfaces of microbes are negatively charged and attract basic dyes – positive staining.
negative staining – microbe repels dye & it stains the background

30. Staining simple stains –one dye is used
differential stains – use a primary stain and a counterstain to distinguish cell types or parts. examples: Gram stain, acid-fast stain and endospore stain
special stains: capsule and flagellar stains

31. Preparing Smears for Staining
Stains consist of a positive and negative ion.
In a basic dye, the chromophore is a cation.
In an acidic dye, the chromophore is an anion.
Staining the background instead of the cell is called negative staining.

32. Staining procedures
Simple stains – use basic dyes to make cells visible
Differential stains – to distinguish between types of microoganisms
1. primary staining
2. Destaining
3. counterstaining
ex) Gram stain (Gram positive and negative bacteria)
Primary stain (Gentian violet)  Mordant (Iodine)  Decolorization (ethanol) 
Counterstain (safranin)
Acid-fast stain (acid-fast bacteria : mycobacteria, some actinomycetes)
Primary stain (Carbolfuchsin)  Mordant (heating with steam)  Decolorization (HCl + ethanol)
 Counterstain (Methylene blue)

34. Prosedur Pengecatan Gram
Pengecatan ini termasuk pengecatan positif-diferensial. Cuplikan inokulum harus diletakkan dalam gelas benda dan difiksasi dengan pemanasan. Beberapa reagen yang diperlukan adalah:
Crystal Violet (Primary Stain)
Iodine (Mordant)
Decolorizer (ethanol)
Safranin (Counterstain)
Aquades (dalam botol semprot)

36. Differential Stains: Gram Stain
Color of
Gram + cells
Gram – cells
Primary stain:
Crystal violet
Purple
Mordant:
Iodine
Decolorizing agent:
Alcohol-acetone
Colorless
Counterstain:
Safranin
Red

47. (Staphylococcus)

48. Figure 3.9a Gram-staining procedure. Steps of the procedure.
Special stains – reveal specific parts of a microbes, such as the wall, the nucleoid, an endospore,
membranes, flagella, or the capsule.
ex) flagella stains
 renders flagella visible by adding a material, making them thicker (tannic acid)
 thickened flagella are stained with dye (rosaniline)
negative stains
 Identify the presence of capsules

52. Differential Stains: Acid-Fast Stain
Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast.
Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with a different color basic stain) to see them.
Figure 3.12

54. Special Stains Negative staining is useful for capsules.
Heat is required to drive a stain into endospores.
Flagella staining requires a mordant to make the flagella wide enough to see.
Figure 3.13a-c