WCU Lab Slides for West Coast Studentsx - Evan Pepper

shamebagΒιοτεχνολογία

22 Φεβ 2013 (πριν από 4 χρόνια και 5 μήνες)

91 εμφανίσεις

Microscopy

Most microorganisms are in the

micrometer

size range


m = 1 meter


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


mm = millimeter = 10
-
3
meters


μ
m = micrometer = 10
-
6

meters


nm = nanometer = 10
-
9

meters


1 Angstrom = 10
-
10

meters


pm = picometer = 10
-
12

meters

Size Comparisons Among Atoms, Molecules, and Microorganisms

Light microscopy

Electron

Microscope

Images

Magnification

vs
.
resolution


Magnification

= increase in apparent size




of an object



Resolution

= ability to distinguish two

objects
as separate from each other

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



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

Gram Stain Procedure

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

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.

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”

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


Culture media

PEA Agar for Gram
-
Positive bacteria

Mannitol Salt agar for
pathogenic
staphylococci

Selective Media

Phenylethanol Agar
, selective for Gram
-
positive organisms.


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



Enriched Media

Neisseria

Gonorrhea


on Chocolate Agar

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




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



Serial dilution

of cultures

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

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

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

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

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

Parasitology


Parasitology = study of protozoa and
multicellular parasites such as


worms, ticks, lice, and fleas



Today
-




Examine microscope slide sets





+



Live “wet mounts”



Coccidiosis


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

Eimeria necatrix

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

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

Eye
-
worm (loa loa)

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

PUBIC LICE (“CRABS”) ON LOCATION !!

www.visualdxhealth.com

Scabies Mites

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

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

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

Using the Spectrophotometer to count bacteria

GO TO


ht瑰:⼯/ww.phy獩捳cc獢sju.edu/stat猯捨i彦it.h瑭t


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 ↑

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)

Biotechnology and Genetic Engineering

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 (helps
anemics
/cancer patients)


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 !

Construction of a Recombinant DNA Molecule

Can we move DNA between these two
organisms ???

Aequorea victoria
(Sea Jelly

)

E. coli

(bacteria)

“THE GENE GUN”

BIOTERRORISM


“THE BIG 6”


ANTHRAX



TULAREMIA


(“rabbit fever”)



PLAGUE



BOTULISM



SMALLPOX



HEMMORHAGIC FEVER
VIRUSES


(EBOLA/MARBURG)

Epidemiology


Study of the incidence, prevalence,
distribution, and control of a disease in a
population.



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)

Food Microbiology: 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………)