CHAPTER 3

OBSERVING MICROORGANISMS THROUGH A MICROSCOPE

Dr. ReitanoSUFFOLK COUNTY COMMUNITY COLLEGE

Microorganisms were first observed by Antonie van ____________, using a ________ microscope. A _____________microscope has only one lens.

Cowan “Microbiology”

RELATIVE SIZE as a TOOL in ________________

Cowan “Microbiology”

_____ System – International System of Units

SOME TYPES of MICROSCOPES• ________

– Bright field• __________ LIGHT

MICROSCOPE

– _____________

• ELECTRON– Transmission– Scanning

________________MICROSCOPY• DISTINGUISHING FEATURES

– Visible light passes through a series of ________ which magnify the specimen and allows fine detail to be observed (___________)

– Specimen appears _______ – Field appears lighter

• PRINCIPLE USES– Common, multi-

purpose microscope– Used to observe ____

specimens and preserved, stained

(___-living) specimens– Provides fair cellular

detail

_________________ MICROSCOPE

Figure 3.1b

___________ Light Microscopy

• In a compound microscope, the image from the ____________ lens is magnified again by the __________ lens

• Total magnification =objective lens ocular lens

PATHWAY of _____

Properties of a Compound Light Microscope: 1. _____________________

Magnification of _________

LensX

Magnification of Ocular

Lens

= Total

Magnification

Low Power ___X 10X 100X

High Dry ___X 10X 400X

Oil Immersion

___X 10X 1000X

2. _____________The ability of a lens system to accurately distinguish between two separate points, that lie close to each other, as separate and distinct.

(structures less than 0.2um cannot be resolved with the compound light microscope)

Figure 3.3

_________ in the Compound Microscope

______ index - a measure of the light-bending ability of a medium, such as air

•Air may bend the light so much that it misses the small size of the opening in the 100x objective lens

•____________ is used to keep light from bending

_______________ MICROSCOPY• DISTINGUISHING

FEATURES– Specimens are stained

with ____________dyes– ultraviolet light is used

which causes ___________ molecules in a specimen to emit light

PRINCIPLE USES Rapid detection and

identification of organisms in tissues

Excellent _____________ tool

ELECTRON MICROSCOPETYPES:

________________________________________________

__________ ELECTRON MICROSCOPY

• DISTINGUISHING FEATURES• Beam of __________(not

light) are reflected from the specimen

• __________dimensional image produced

• Magnification 1,000 to 10,000x

• Principle Uses• Observing _______

details of cells and viruses

___________ ELECTRON MICROSCOPY

• DISTINGUISHING FEATURES– Beam of __________ (not

light) pass through the specimen

– ___ dimensional image is produced

– Magnification 10,000 to 100,000x

PRINCIPLE USESPRINCIPLE USES Examination of _______ _____________ of cells

COMPARISON of _____ MICROSCOPES and ____________ MICROSCOPES

Cowan and Talaro

MICROSCOPIC OBSERVATION• TWO TYPES of SPECIMENS:

– 1. ____________• Wet Preps, Wet Mounts.• Living organisms suspended in fluid-organisms have

________________with surrounding fluid.• Used to study: size, shape, arrangement of cells,

(morphology), __________ , and _________.– 2. STAINED

• Fixed Smear Preparations.• ________________ organisms.• Contrast is created to allow cellular characteristics to

stand out.• Used to study size, shape, arrangement of cells

(morphology) but not motility.

PREPARATION of _______ SMEAR

_________ STAINING

Nester et al.

SMEAR: A ___ film of a solution of ________ on a slide A smear is usually ____ to ______ the microbes to the slide and to ______ the microbes. ______: Coloring the microbe with a ____ that emphasizes certain structures

VARIOUS STAINING CATEGORIESACIDIC STAINS

• __________ charge

(negative stain)• _________ by cells• _________ is stained• Ex.: India ink ________

stain

BASIC STAINS• ________ charge (positive stain)• ________ to cells (cells

have a _______ charge)• Cells are stained• Ex.: Methylene Blue, Crystal Violet, Safranin, Malachite Green

VARIOUS STAINING CATEGORIES cntd.SIMPLE

_____ dye Simple procedure Ex.:

• Methylene Blue

• Crystal Violet

• Safranin• Malachite

Green

DIFFERENTIAL Two dyes

__________ __________

Contrast 2 cell types or parts

Complex procedure

Ex.: _____ Stain ___________

Stain

SPECIAL Targets

_______ cell _______ Such as:

capsules flagella

spores Ex.:

India Ink Flagella Stain Spore Stain

_______________ STAIN_______________ STAINGRAMGRAM STAINSTAIN

• Developed by Dr. Hans Christian Gram in 1884• Most widely used procedure for staining ___________ • Classifies bacteria into two groups

– Based on differences in _________ STRUCTURE

Gram positive Gram negative

Basic _____ of Most Bacterial Cell _____

determine _______provide structural _________

Basic _______ of Bacterial Cell Walls:

COMPARISON of GRAM POSITIVE and GRAM NEGATIVE BACTERIA

GRAM ___________ BACTERIA

Ex.: Staphylococcus aureus Streptococcus

pyogenes

GRAM _________ BACTERIA

Ex.: Escherichia coli Klebsiella pneumoniae

Nester

STEPS:

Color of Gram-positive bacterial cells

Color ofGram-negative bacterial cells

1. ________ stain: Crystal Violet

Purple Purple

2. ____________: Iodine

Purple Purple

3. _____________ agent:** Acetone Alcohol

Purple _________

4. Counterstain: _____________

___________ ________

GRAM STAIN PROCEDURE

_______ STAINING PROCEDURE

Tortora

________________ STAINAcid Fast Stain

• Used for bacteria with _______ material in cell wall

• Several procedures*• Ex.:

– Mycobacteria species– Nocardia species

_____bacteria species

Nester

DIFFERENTIAL STAIN________________

• Used for bacteria with waxy, lipid (_____ ______) material in cell wall

• Several procedures*1. Primary stain: Carbolfuchsin 2. Decolorizer: _____ Alcohol3. Counter stain: Methylene blue

• Ex.:– Mycobacteria species– Nocardia species

Mycobacteria species

Nester

STEPS:

Color of ___________

Bacteria(Mycobacteria sp.)

Color ofNon–Acid-

fastBacteria

1. Primary stain: __________

Red Red

2.__________ agent:** _____-alcohol

Red ___________

3. Counter-stain: _____________

____ ______

___________ STAIN PROCEDURE

ACID FAST STAIN Acid-fast staining of a patient’s

________ is a rapid, reliable, and inexpensive method to diagnose __________. What is the genus and species of this organism?

This is an acid-fast stain of a patient’s _______. What is the disease associated with this organism?

SPECIAL STAINSUsed to distinguish _____ of cells

• CAPSULE

______________

ENDOSPORE

Tortora

COMPARISON of STAINS

Cowan et al.

SOME IMPORTANT STAINS USED in MICROSCOPY

________

- Methylene Blue

- Carbolfuchsin

- Crystal Violet

- Safranin

DIFFERENTIAL

- Gram

- Acid-Fast

SPECIAL

- __________

(Negative,

Acidic)

- Endospore

- Flagella