Chapter 3 Microbiology

Anton Van Leeuwenhoek

first to observe live microorganisms
known as the father of microbiology
made improvements to the 1st microscope and got 300X magnification

most of pathogenic fungi are

dimorphic

micrometer

is equal to 0.000001m

micro indicates that the unit following should be divided by 1 million

nanometer

is equal to 0.000000001m

1 meter equals how many centimeters

100 cm

1 metere equals how many micrometers

1000 micrometers

1 micrometer equals how many nanometers

1000 nanometers

Size of Protozoa

100 micrometers

Size of Yeast

8 micrometers

Size of Bacteria

1-5 micrometers
(some are much longer than wide)

Rickettsia

0.4 micrometers
intracellular bacterium, gram negative

Chlamydia

0.25 micrometers
causes syphillis

Mycoplasma

0.25 micrometers
gram negative
causes variety of pnemonia, urinary tract infections

bacteria contains both

DNA and RNA

Viruses contain

DNA OR RNA
never both

compound light microscope

has a series of lenses and uses visible light to observe specimens
uses white light

magnification

ocular lenses and objective lenses

resolution

AKA resolving power
is the ability of the lens to distinguish fine detail and sructure

Resolution for a light Microscope

0.2 micrometers

Contrast

stains change refractive index

refractive index

a measure of the light bending ability of a medium

as you go up in magnification, what do you need to adjust

light, need more of it and adjust it by using substage iris diaphgram lever

as magnification goes up, what happens to the lenses

they get smaller which is why we have to add more light

immersion oil

has same refractive index as glass
helps us preserve the direction of light rays at the highest magnification

brightfield microscope

what we use, simplest of all optical microscope illumination
dark objects appear against a bright background

darkfield microscope

used to examine live microorganisms, objects are visible against a dark background

What microscope should you use to view spirochetes?

darkfield microscope

spirochete

a corkscrew shaped bacterium with axial filaments, type of spirilla bacteria

Treponema pallidum

spirochete that causes syphillis

Borrelia burgdorferi

spirochete that causes lyme disease

Fluorescence Microscope

absorbs UV light and emits visible light, used a lot in clinical labs

immunofluorescence

a diagnostic tool using antibodies labeled with fluorochromes and viewed through a fluorescence microscope

the smaller the wavelength

the higher the resolution

prokaryotes

have no cell organelles

electron microscope

uses electrons and electromagnetic lenses to view specimens, there are 2 types

2 types of electron microscopes

scanning electron
transmission electron

transmission electron microscope

a electron microscope that provides high magnification 10,000-100,000X of thin sections of a specimen

scanning electron microscope

an electron microscope that provides 3D views of a specimen magnified 1000-10,000X

staining

simply means to color the microorganism with a dye that emphasizes certain structures

fixing

in slide prep: the process of attaching the specimen to the slide

smear

thin film of material containing microorganisms spread over the surface of a slide

Technique for preparing a slide for light microscopy

1) Smear
2) Air Dry
3) Heat Fix

Basic Dyes

cationic chromophore
(positive charged)

Acidic Dyes

Anionic Chromophore
(negative charged)

Chromophore

stains are salts composed of positive and negative ions, one of which gets colored called a

Negative Staining

a procedure that results in a colorless bacteria against a stained background
the bacteria repels the stain

3 types of staining techniques

simple
differential
special

simple stain

uses a single basic dye and sometimes a mordant

mordant

substance added to a staining solution to make it stain more intensely

type of mordant we will commonly use

iodine

differential stains

twp types
react differently with different types of bacteria and can be used to distinguish them

types of differential stains

acid fast stain
gram stain

Differential Stains use what type of dye?

basic dye

gram stain

differential stain that classifies bacteria into 2 groups: gram positive and gram negative

gram positive

bacteria that retain the color of the primary stain after the alcohol attempts to decolorize it

stains a dark purple or violet

gram negative

bacteria that loses the dark violet or purple color after decolorization

stains pink

Steps to make a gram stain

1) heat fixed smear is covered with a primary stain, a basic purple dye
2) Dye is washed off, mordant is added
3) Slide is washed with a decolorizing agent
4) Alcohol is rinsed off and stained with a basic red dye

negative staining is used for

bacteria with capsules, which is a slime layer around the bacteria

Acid Fast Stain

a differential stain used to identify bacteria that are not decolorized by acid-alcohol

cells that retain a basic stain in the presence of acid alcohol

which dye can be used as a simple stain?

any basic dye
usually methylene blue, safranin, crystal violet

Acid Fast Stains are used to identify what type of bacteria

myobacterium

Special Stains

used to color and isolate specific parts of microorganisms

Negative staining for capsules

uses basic stain and negative stain to test for the presence of a capsule

flagella staining

requires a mordant to make the flagella wide

endospore stain

heat is required to drive the stain into it