Cellular makeup Prokaryote Eukaryote Subgroups of Microorganisms Table

Cellular makeup Prokaryote Eukaryote

Subgroups of Microorganisms Table 1. 2

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

Struktur organel sel Eukaryote (Starr, 1998)

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

compound light microscope

Pathway of light

Effect of wavelength on resolution

Effect of magnification

Oil immersion lens

• magnification – ability to enlarge objects • resolving power – ability to show detail

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.

Bacterial shapes and arrangements

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

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…. .

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

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

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

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.

Staining procedures • Simple stains • 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) – use basic dyes to make cells visible Acid-fast stain (acid-fast bacteria : mycobacteria, some actinomycetes) Primary stain (Carbolfuchsin) Mordant (heating with steam) Decolorization (HCl + ethanol) Counterstain (Methylene blue)

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)

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

(Staphylococcus)

Figure 3. 9 a 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

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

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. 13 a-c