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Microscopy

Most microorganisms are in

the micrometer size range

• m = 1 meter

• cm = centimeter = 1/100m = 10-2 meters

• mm = millimeter = 10-3 meters

• μm = micrometer = 10-6 meters

• nm = nanometer = 10-9 meters

• 1 Angstrom = 10-10 meters

• pm = picometer = 10-12 meters

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Size Comparisons Among Atoms, Molecules, and Microorganisms

FISH

TAPEWORM

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Light microscopy

Magnification vs. resolution

• Magnification = increase in apparent size

of an object

• Resolution = ability to distinguish two

objects as separate from each other

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Light microscopy has other

optical configurations

• Dark-field microscopy

• Phase-contrast microscopy

• Fluorescence microscopy

Flourescence Microscopy Image

Fluorescence microscopy of endothelial cells using three labels. Red labels the mitochondria, green

labels the F-actin cytoskeleton and blue labels the nucleus. Image by Steve Karl

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Electron Microscopy

Electron Microscope

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Electron

Microscope

Images

Streptococcus pneumoniae

Gram-positive diplococci with capsule (haloe) formation,

located outside neutrophils.

http://www.fujita-hu.ac.jp/~tsutsumi/case/case071.htm

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Legionella pneumophila

As seen in the cytoplasm of macrophages

Anthrax bacterium (Bacillus anthracis) with human blood cells.

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Pseudomonas aeruginosa

Gram-negative rods floating within mucoid matrices.

Bordetella pertussis

Mainly seen here outside neutrophils

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Bacteria: Staining Techniques

• Positive Stain (basic)

• Negative Stain (acidic)

• Gram Stain

• Acid-Fast Stain

• Capsule Stain

• Spore Stain

• Flagella Stain

Why Stain ???

• A) Achieve Contrast

• B) View Size, Shape, + Cellular Structures

(cell wall, flagella, glycocalyx, spores, etc.)

• C) Classify/Partially identify organisms

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Staining: Smear Preparation

• Smear = a slide with microbes on it, ready to be stained

1) Label slide

2) Add water drop to the slide

3) Add the microbe to the water drop

4) Air-dry 5-10 minutes

5) Heat-fix (basic stains only, not acidic stains or the capsule stain)

Simple Staining Reactions in Microbiology

Positive Stain

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Positive Stain

Typical Bacillus stained with Crystal Violet

Negative Stains

Bacillus stained with negrosin

Cocci stained with Negrosin

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Gram Stain Procedure

Streptococcus mutans

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E. coli gram stain

Gram Stain

• 2 slides/group:

• 1 bacterium (tube)

• 1 gum-line sample

• Procedure:

• Make Smear

• Heat-Fix

• Gram-Stain

• View with Microscope

• Materials • Gloves

• Slides

• Pen

• Loop

• Toothpick

• Sparker/Bunsen Burner

• Bacteria

• Test Tube Rack

• Clothespin

• Staining Kit

• Transfer Pipette

• Drying/Bibulous Paper

• Microscope/Oil/Lens Paper

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Bacillus cereus with neutrophils

Acid-fast Stain

Mycobacterium (acid-fast positive)

Designed to identify Mycobacteria

-- Mycobacterium tuberculosis

-- Mycobacterium leprae

Mycobacteria have a special wax

layer in their cell wall (made of

mycolic acid)

Wax helps these bacteria to resist

acid-alcohol de-staining step

(“acid-fast” = have ability to retain

the primary stain in spite of acid-

alcohol treatment)

Can be used on sputum

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Capsule Stain

Klebsiella Pneumonia

Capsule = Glycocalyx

-- sticky layer around

some bacteria

-- helps them to retain

water, attach to tissues,

and avoid the immune

system

COMBINATION STAIN: two stains on top of each other; one

is acidic (stains background), other is basic (stains the cell);

capsule resists both stains and appears as a white “halo”

around cells.

Bacteria from a dirty dish; 1600x, capsule

stain (negrosin then safranin)

http://picasaweb.google.com/marc.murison/BestMicro/photo#5114441791829109154

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Spore Stain

Resistant structures formed by

some bacterial species

Examples: bacteria that cause

anthrax, botulism, tetanus,

gangrene, diarrhea (“C. diff.”)

Difficult to stain, need to use

steam and lots of stain to

visualize them

Can have “endospores” or

“free spores”

Anthrax spore stain

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Flagella Stain

Also go to wet mount video at http://www-micro.msb.le.ac.uk/video/motility.html

Provide motility (movement)

-Long, thin proteins that are

fragile, break easily

-Difficult to stain and visualize

-Other methods exist to look at

motility (wet mount technique)

Bacteria: Culturing and Counting Techniques

• How to grow microbes: Types of media

• How to isolate microbes: Throat swab / “Streak” plate

• How to count microbes: Serial dilution / “Spread” plate

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Culture media

PEA Agar for Gram-

Positive bacteria

Mannitol Salt agar

for pathogenic

staphylococci

Selective Media

Phenylethanol Agar, selective for Gram-positive organisms.

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Differential Media Example

Mannitol Salt Agar

Used when Trying to examine “Staph” bacteria

S. Aureus – potential pathogen

S. Epidermidis – harmless resident of skin

Plate contains a dye that turns yellow at low pH

(if the bacteria are producing acid)

S. Aureus can eat the sugars in the media

(mannitol) and produces acid as a “waste”

product

S. Epidermidis cannot eat the sugar at all

Selective and Differential: McConkey Agar

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Enriched Media

Neisseria Gonorrhea

on Chocolate Agar

THIS IS

EMB AGAR (Eosin-Methylene Blue)

IT CONTAINS DYES THAT INHIBIT

GRAM POSITIVE BACTERIA.

IT CONTAINS LACTOSE THAT ALLOWS

LACTOSE-FERMENTERS TO HAVE A

GREEN/METALLIC COLOR

THIS IS AN EXAMPLE OF which type of

media:

A. SELECTIVE

B. DIFFERENTIAL

C. ENRICHED

D. ALL OF THE ABOVE

E. A AND B

F. B AND C

G. I DON’T KNOW

CHECK YOUR

UNDERSTANDING

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Streak Plate Technique

Streak Isolation on Nutrient Agar

GOAL: separate different

bacterial species from

each other when they are

in a mixture

ISOLATION of colonies:

a colony represents a

single bacterium and its

overnight descendants

Materials needed: Gloves/swab/loop/tongue depressor/plate/sparker/alcohol/tape/pen

Hemolysis

Alpha, Beta, and Gamma hemolysis

Alpha = partial

breakdown of the

red blood cells

(greening)

Beta – total

destruction of

RBCs

(white/clear zone)

Gamma – no

destruction of

RBCs

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http://gold.aecom.yu.edu/id/micro/hemolysisabg-72.jpg

Hemolysis

Serial dilution of cultures

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Biochemical Tests

• Bacteria and other microbes can be

classified/identified according to the

types of enzymes they possess

{and thus the types of biochemical

reactions they can perform}.

Catalase Test

Staphylococcus

aureus

Enterococcus

faecalis

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COAGULASE TEST

An enzyme produced by

some, but not all, bacteria

Positive reaction = clump or

clot formation in the media

within 2-6 hours

Negative reaction – no clot

Media is rabbit plasma broth

Makes bacs more dangerous

because unwanted clots are

produced and the clot itself

shields them from phagocytes

UREASE TEST

An enzyme produced by

some, but not all, bacteria

Urea – a toxic compound,

kills bacteria (in stomach, in

bladder, kidneys, etc.)

Some bacteria can break

down urea to carbon

dioxide and ammonia

(basic, can neutralize

stomach acid)

H. pylori is urease +

Dye is pink

when pH is

basic

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OXIDASE TEST

CITRATE TEST

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INDOLE TEST

BILE ESCULIN AGAR

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Dichotomous key

• a map for the identification of organisms

based on a series of choices between

alternative characters

• can be stains, biochemical tests,

antibiotic susceptibility, or other

Dichotomous Key -- a simple example

--------------------------------------

--------------------------------------

--------------------------------------

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Partially complete example…..

ALL 13 Organisms

Gram + Gram -

Cocci,

clusters

ML, SE

Cocci,

Chains

EF

Large

Rods

BC, BM

Normal Rods

LP, BS, BP

Rods

PF, CV

Short Rods

EC, KP, SM

SE ML

Lactose

F -

Glucose

F -

BC BM

FG F -

BP LP BS

Lactose Glucose

F -

CV PF

Indole

+ -

EC KP SM

motility

SM KP

+ -

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API-20E kit example (A)

{Patient 1 symptoms: severe abdominal

cramps and watery diarrhea. There is little or

no fever, and no vomiting.

Culture ID #8101

Identification:

Escherichia

coli

5144572

culture

no.

O

N

P

G

A

D

H

L

D

C

O

D

C

C

I

T

H

2

S

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G

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L

G

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A

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O

S

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R

H

A

S

A

C

M

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L

A

M

Y

A

R

A

8101 + – + + – – – – + – – + + – + + + + – +

Example Data Table

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API-20E kit example (B)

Culture ID: 8P14

Patient Symptoms:

PAIN, fever, diarrhea

and abdominal cramps

culture

no.

O

N

P

G

A

D

H

L

D

C

O

D

C

C

I

T

H

2

S

U

R

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T

D

A

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P

G

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L

G

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A

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C

M

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A

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A

R

A

8P14 – – + + – + – – – – – + + – + + – + – +

Identification:

Salmonella

sp.

Example Data Table

7-digit ID code = 4501552

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Parasitology

• Parasitology = study of protozoa

and multicellular parasites such as

worms, ticks, lice, and fleas

• Today -

Examine microscope slide sets

Live “wet mounts” and worm dissection

Introduction: Coccidiosis

http://www.anri.barc.usda.gov/pbel/images/bigchicklittlechick.jpg

Eimeria necatrix

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Trypanosomes: African Sleeping Sickness

http://www.med.uni-marburg.de/stpg/ukm/lt/hygiene/schwarz/Trypanosoma.jpg

LIFE CYCLE

OF TSE-TSE

FLY =

VECTOR

Balantidiasis

Balantidium coli

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Entamoeba histolytica

http://www.weizmann.ac.il/Biological_Chemistry/images/mirelman.jpg

http://www.microscope-microscope.org/applications/pond-critters/protozoans/sarcodina/entamoeba.htm

Malaria

http://bepast.org/docs/photos/malaria/Malaria.jpg

Plasmodium spp.

Female anopheles mosquito

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Eye-worm (loa loa)

http://maven.smith.edu/~sawlab/fgn/pnb/loaloa.html

PUBIC LICE (“CRABS”)

www.visualdxhealth.com

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Scabies Mites

http://www.stanford.edu/class/humbio103/ParaSites2004/Scabies/scabies1.jpg

Parasite Lab

Materials

1.Black Box – Protozoa Slides

2.White Box – Multicellular Parasite Slides

3.Green Jar – Preserved worms (for inspection)

4.White Jar – Preserved worms (for DISSECTION)

Precautions/Safety –

-- HANDLE SPECIMENS WITH FORCEPS AND GLOVES

(+USE GOGGLES DURING DISSECTION)

-- DISPOSE DISSECTED WORMS IN BIOHAZARD BIN

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Parasite Lab – Week 5 Microbiology

Dissection

-- Use your dissecting tools to make a longitudinal cut in the

roundworm provided (Ascaris). Try to distinguish if your

worm is male or female (see photo below). Note that the

major structures you see will be used for sexual

reproduction and digestion.

Case Study: Chagas Disease

Gross anatomy of a heart that has

been damaged by chronic Chagas

disease

Trypanosoma cruzi

(causative agent)

Reduviid “Kissing”

Bug (Vector)

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Top: A pork tapeworm (Taenia solium)

cysticercus, the form in which the

tapeworm is found in an infected brain.

(Colorized image by P. W. Pappas and S.

M. Wardrop, courtesy of P. W. Pappas,

Ohio State University.)

Bottom: T. solium cysticerci in the brain

of a nine-year-old girl who died during

cerebrospinal fluid extraction to diagnose

her headaches. This was in the 1970s—if

it had happened 10 years later,

noninvasive computerized tomography

would have given an accurate diagnosis,

and the parasites could have been killed

with drugs. (Image courtesy of Dr. Ana

Flisser, National Autonomous University

of Mexico.)

PORK TAPEWORM

CYSTS IN THE BRAIN

Wet Mount Technique

• Method to visualize living microbes

• Uses a cover glass and “depression” slide

• Also known as the “hanging drop” technique

Planaria (flatworm) Trichomonas vaginalis

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Control of Microbes

Measuring Zones of Inhibition

Antibiotic Disc Diffusion Assay

Pour 25mL agar plates

Grow bacteria in liquid culture

to 100,000,000/ml

Spread 150 microliters on plate

Add antibiotic discs to plate

Let bacteria grow overnight

Measure ZOI and compare to

standard table

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Using the Spectrophotometer to count bacteria

GO TO http://www.physics.csbsju.edu/stats/chi_fit.html

Absorbance is

proportional to

number of bacteria

Salt

• Used to preserve foods (meats/fish/etc.)

• Works by dehydrating microbes -- (lose water, shrivel)

• Creates hypertonic environment (re: osmotic stress)

• Exception: Halophiles prefer 3% NaCl or ↑

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pH

• Measures H+ ion concentration

• ↑ H+ means more acidic (lower pH),

• ↓ H+ means more basic (higher pH)

• Most microbes are neutralphiles (5.5-8.5)

• Some are acidophiles (<5.5)

• A few are basophiles (>8.5)

• Examples: “pickling” with vinegar (acid) or basify shampoos

Filtration

AcetatePlus VP vacuum filtration units with cellulose acetate

•Method to physically trap

microbes

•Used to purify liquids/air

•Has tiny holes called “pores”

(anything larger than the pore size

gets trapped on the filter itself)

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Biotechnology and Genetic Engineering

Genetic engineering

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Restriction Enzymes and Recombinant DNA

Construction of a Recombinant DNA Molecule

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Human genes can be cloned in bacteria

“Artificial” Transformation

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Examples of products made by recombinant DNA technology

1. HUMALOG – Human insulin made by E. coli bacteria

2. PROCRIT – Human erythropoietin made by mouse cells

3. NEUPOGEN – helps humans grow more neutrophils,

made by inserting human DNA into E. coli

4. RECOMBIVAX – Hepatitis B vaccine made by inserting

viral DNA into yeast cells and growing up viral proteins !

Applications of Genetic engineering

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The Genetic Code

Bt crop concerns

http://www.biology-blog.com/images/blogs/10-2007/genetically-engineered-corn.jpg

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Golden Rice

“THE GENE GUN”

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Can we move DNA between these

two organisms ???

Aequorea victoria (Sea Jelly ) E. coli (bacteria)

The pGLO plasmid

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THE

PROCEDURE

Expected Results

LB/AMP/pGLO LB/AMP/ARA/pGLO LB/AMP LB

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Transformed Bacteria

GFP Fly

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GFP mice and RFP cat

Oinky Oinky….

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Fun on a plate

Living bacteria expressing 8 different colors of fluorescent proteins.

BIOTERRORISM – “THE BIG 6”

• ANTHRAX

• TULAREMIA

(“rabbit fever”)

• PLAGUE

• BOTULISM

• SMALLPOX

• HEMMORHAGIC

FEVER VIRUSES

(EBOLA/MARBURG)

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Epidemiology

• Study of disease “determinants” in populations

(infectious, environmental, genetic, and lifestyle)

• Includes measurements of incidence,

prevalence, distribution, and control of diseases

• Usually involves collecting and analyzing data

(heavy statistics!).

Epidemiological terms

• Epidemic: An outbreak of disease that attacks a large percentage of the population simultaneously and may spread through one or several communities.

• Pandemic: When an epidemic spreads throughout the world.

• Endemic: a disease that exists permanently in a particular region or population. Usually a small percentage of persons are affected.

• Outbreak: a short epidemic (contained)

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Smoking vs. lung cancer per capita per year (country)

Hungary 2515

Japan 2510

USA 2020

South Africa 1950

UK 1700

France 1690

USSR 1650

Brazil 1200

Philipines 1150

Venezuela 950

Zaire 150

India 100

The Oxford Atlas of the World, ISBN 0-19-520955-9, published in 1992

From Parkin, D. M. et al. CA Cancer J Clin 2005;55:74-108.

Extras

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Genetic Vaccine Questions

• What are some of the disadvantages of inactivated, subunit, and attenuated viral vaccines?

• What are the potential advantages of genetic vaccines?

• In what genetic form and how are genetic vaccines delivered to the body cells?

• How can the positive effects of the vaccines be amplified/increased ?

• What human genetic vaccine tests are currently being performed/attempted?

RNA interference questions

1. How is RNA interference more precise than the interferon response?

2. What are some of the ultimate goals of “directed” RNA interference ?

3. What type of RNA proved most useful in RNAi, single or double

stranded RNA ??

4. What are siRNAs? MicroRNAs?

5. How does RNA interference help in learning about the functions of

genes ?

6. What is the most difficult challenge facing human RNAi therapies ?

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Making Yogurt

• Heat milk (450ml) to 83°C while stirring

• Allow to cool to 43°C degrees

• Put starter culture (1/2 cup) in separate mixing bowl

• Slowly add milk to starter culture w/ stirring

• Cover with foil and punch holes in foil

• Incubate 2-6 hours at 30°C

• Add fruit (optional)

• Try it (if you are brave………)

Questions for Food Poisoning Film

• Who is getting sick, and why ??

• Where did physical control of microbes

break down ???

• Why do some victims recover quickly,

while others take 10+ years ???