Section 3 Basic Skills.knowledge - haspi

muskrateurekaBiotechnology

Feb 12, 2013 (4 years and 6 months ago)

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Basic Skills and knowledge

for Microbiology and Biotechnology

1)

Measurement
-

using tools correctly

a)

Accuracy and Precision

i)

Accuracy

is the ability to measure to a standard
-


ii)

Precision

is the ability to create the same measure over and over
without variation

iii)


Instruments are
calibrated
so they are accurate; when you use
them correctly, your measurements are precise.

b)

Microscope



a microscope is an instrument that uses lenses to
magnify the image of an object

i)

Magnifying glass
-

simple microscope (one lens)

ii)

L
ight microscope


compound microscope (two
lens)

(1)

www.udel.edu/scope


(2)


Path of light through
microscope

(3)

Inversio
n


As a
result of light waves
being bent as go through the lens, the
image at the eye piece is upside

down
and backwards

(4)

Resolution

-

the ability to tell two objects apart. The better
the resolution, the clearer the picture (refer to TV news where
face of alleged criminal is pixelated to prevent identify).

(5)

Powers of magnification



the times larger an i
mage is than the
original object. In simple microscopes, it is the power of the
lens (2X is twice as large as real object). In a compound
microscope, there are two lenses so the total magnification is
the product of the powers of the two separate lenses.


Ocular Lens

Objective
Lens

Total
Magnification

called

10X

4X

40X

scanning (low)Power

10X

10X

100X

medium power

10X

40X

400X

high dry power

10X

100X

1000X

High oil (must use oil)


c)

Pipet

An instrument made of a glass tube and a bulb that can
measure
or

transfer precise volumes of a liquid by drawing the liquid up into
the tube
.

d)

Micropipet



an instrument that
can measure or transfer precise
very
small volumes of a liquid


usually less than 1 millileter.

e)

Scale/balance
-

An instrument can measure precis
e masses of a liquid
or solid.

f)

Graduated cylinders

-
An instrument made of a glass cylinder can
measure
precise volumes of a liquid.

g)

Note:
Beakers

and
flasks

are NOT calibrated so they are NOT used
for measuring. Observe side of beaker/flask “+/
-

5%”
-

this means
the numbers on the glassware can be off as much as 5%

2)

Solutions and Media


solutions are
homogeneous mixture

of two or more
substances (liquid and liquid, liquid and solid, liquid and gas)

a)

Solutions

i)

Terms



(1)

Solvent



liquid into which somet
hing is dissolved

(2)


Solute



the solid that is dissolved in the solvent (if two liquids,
the one in greater amount is the solvent)

ii)

Percent

(1)

A solution that is based on the amount of solute divided by the
total volume of the solution. In example below, solve
nt is water

(a)


Mass/ volume (read as mass per volume)
-

5g of NaCl in
1O0 mls final volume is a 5% solution. (note


100 mls final
volume, NOT added to 100 mls. Why?)

(b)


mass/mass


5g of NaCl in 100g solution is a 5% solution

(c)

volume/volume


10 ml of Ethano
l in 100 mls final volume is a
10% solution

b)

Molar



a molar solution is one mole in a final volume of 1 liter solution.

i)

a mole (6.023 X 10
23

molecules



Avogodro’s number
) is a ‘chemist’s
dozen’


if you have a mole of molecules you have 6.023 X 10
23

-

ju
st
like if you had a dozen molecules you’d have 12.

ii)

A mole is based on the number of particles

(1)

so a mole of one particle type can has a different mass than
another

(a)

Think of this: if you had a dozen pingpong balls and a dozen
bowling balls, you’d have 12
of each BUT if you weighed
them, the 12 bowling balls would weigh a lot more than the
12 ping pong balls. So …… if you have a mole of pingpong
balls and a mole of bowling balls, which would weigh more?

(b)

Now let’s look at molecules


let’s take water (H
2
O
) and
carbon dioxide (CO
2
). Do you remember how to find the
formula (molecular) weight? Add up the weights of the
atoms in the formula


water is 18 (2 H at 1, one O at 16) and
Carbon dioxide is 44 (1C at 12, 2 O at 16). Here’s the cool
part


Avogadro dis
covered that 1 mole of molecules =6.023
X 10
23

molecules = 1 formula weight in grams for that
molecule.

This means 1 Mole of water weighs 18g, 1 mole of carbon dioxide is
44g!

What is the weight on 1 mole of sodium chloride
-

NaCl?
Yes, 58g!


So if y
ou want a 1 molar solution of NaCl, you dissolve 1 mole
of NaCl in a final volume of one liter water


or 58g

What if you wanted a .5M NaCl solution?

c)

Media



Living things require nutrients. Different types of living
things require different types of nutr
ients. Plants require CO
2
, H
2
O,
and sunlight


inorganic nutrients. Lions are carnivores and require
meat. Cows are Herbivores and need grasses. People are omnivores
and eat their nutrients in both plant and animals form. Do you
remember
autotrophs

and
h
eterotrophs
?


Bacteria
can be either autotrophs or heterotrophs. Autotrophic
bacteria are cyan bacteria


sometimes mistakenly called algae. Most
bacteria are heterotrophs. Some live by absorbing nutrients from
dead materials (decomposers), some from live
materials


the bacteria
that give you zits, for instance. Some live off dead material inside you
-

the
E.coli

that are found in your gut


but the living conditions are a
trade


they produce Vitamin K that you absorb.


Could you identify a virus as an
autotroph or heterotroph?


When we want to grow bacteria in a laboratory


not in their natural
environment, we must supply them with not only the nutrients that
need to grow and reproduce, but also the correct environmental
conditions


temperature, pH, s
alt concentrations. Since bacteria live
in and on their food, the food also provides the pH and salt
concentrations. There are two basic type of food….

i)

Broth


a nutrient mixture in liquid form.

ii)

Solid (agar)


nutrient mixture that has base added


usuall
y agar


that allows the media to solidify. Agar is usually used because it is
not a nutrient for most bacteria


so it doesn’t change the nutrient
content of the media. Gelatin is sometimes also used to solidify
media, but it is a protein source for most

bacteria.

iii)

Petri Dishes are usually filled with agar media that is 1 to 1.5%
agar. (So, how many grams of agar are used to make 100 mls of
agar media? Why is this not a molar solution?)

iv)

Motility agar


is less dense (why?)


is usually 0.5% agar.

v)

Agar i
s a polysaccharide


purified from Brown Kelp
-

the same
stuff you see on the beaches. A similar agar is used in Japanese
foods (agar
-
agar) and ice cream as a thickener.

(1)


Powdered agar is yellow in color; a long stringy
molecule that looks like a balled u
p piece of
string


(2)

When agar is added to liquid and heated, the
polysaccharide molecules move into long strings,
and then as they cool, they reform the ball, but because they
are in solution, they curl around each other. With a large
number of mole
cules (1 to 1.5%) the agar becomes relatively
solid. Lower numbers of molecules means the agar is softer
(0.5%).

(3)

Agarose, which we will talk about later during electrophoresis,
is a highly purified form of agar. It is pure white.

d)

Solution



There are some

solutions that are made in small amounts


usually because the solute is either very expensive, or not stable in
solution (meaning you can’t store it so making a lot is wasteful.) In
microbiology and biotechnology, the two most common of these are
enzyme
solutions and antibiotic solutions. They are usually made in
mg/ml
.

i)

Enzymes are proteins that have a catalytic function


they speed
up specific chemical reactions. Examples are restriction enzymes
that cut DNA or ligase that can make the sugar
-
phosphate b
onds
and put the DNA back together. As proteins, they are made in
solvents that have the optimum pH and salt concentrations. Very
little of the enzyme solution is used when setting up reactions
because enzyme molecules can work over and over and over witho
ut
damage.

ii)

Antibiotics


antibiotics are substances that kill bacteria.
Antibiotics can be purchased in powdered or liquid form.
Antibiotics are usually added directly to the agar or broth when
the temperature is approximately 40

C. If the temperature
hott
er, it can damage the antibiotics so they don’t function.

3)

Growing/Counting Bacteria
-

Bacteria grow fast


they can reproduce
every 20 minutes under optimum conditions. To study bacteria or to use
them to produce products, scientists need to isolate pure b
acterial
strains


a culture that has only one type of bacteria in it, and sometimes
ever harder, they need to keep that strain pure as they work with it.
Contamination by unwanted microorganisms is very common. So remember
this rule when working with bact
eria:


Just because it looks clean doesn’t mean it’s sterile

The corollary: What you can’t see CAN hurt you


or at least your culture.

A few terms may help here:

Sterile



means without life of any kind

Disinfected



means to reduce or inhibit growth of o
rganisms

a)

Sterile technique

i)

http://www.mc.uky.edu/oaa/curriculum/iid98/manual/00labtechniq
ues.htm


ii)


Also see PDF Sterile Technique

iii)

Sterile technique, sometimes called ase
ptic technique, is a way to
handle materials in the laboratory that prevent any microorganisms
from the environment from contaminating your cultures. A
technique is a
method

of performance. It is a way to do things to
ensure no outside microorganisms ent
er your cultures. Some parts
of sterile technique are easy


like autoclaving materials and
equipment to make sure they are sterile. Some parts are harder


like transferring a pure culture from one tube or plate to another.
Sterile technique is hard only
because there are so many details to
keep track of
-

but the key is to be methodical and prepare ahead
of time. Take your time and make sure your equipment and area
are ready and it will save you hours of trying to re
-
isolate your
culture.

b)

Transfer of c
ultures



since bacteria are alive, they will run out of
food (or their metabolism will create waste products and foul their
environment), so to keep your cultures healthy, they must be
periodically moved to new media. This usually involves transferring to

like media (liquid to liquid, slant to slant, plate to plate) using sterile
technique.

c)

Liquid cultures


liquid cultures are used to grow many bacteria at a
time. As the number of bacteria in liquid media increase, the liquid
will become cloudy.

d)

Plat
es for colonies



if you want to see the structure of a colony of
bacteria, or you want to start a liquid culture with a group of bacteria
you know are all the same, you will plate out bacteria on agar plates.
Here the media is solid, and the bacteria sit
on top. In a spreading
technique, you pull bacteria across the plate using an inoculating loop,
until individual bacteria are rubbed off the loop. By incubating these
plates, the bacteria start to grow. Since the media is solid, the
bacteria cannot float a
way from each other as they do in liquid; rather
they pile up. When there are about a million bacteria, this pile gets
large enough to see with the naked eye and we call this a colony. Each
colony started from a single bacterium, so each colony is a clone



genetically identical can be used to start a pure culture. (Note: It will
be a bit different for viruses, but we’ll talk about that when we do it)

e)

Spreading Plates


If you have liquid cultures and want to grow
bacteria across the entire surface of th
e plate (called a lawn), then
the drop of bacteria are placed in the center of a plate and a
sterilized glass spreader (“hockey stick”) is used to spread the
solution across the entire plate. In this technique, there are so many
bacteria next to each other

that you cannot see the individual
colonies, so it is called a lawn


just like if you look at a lawn, you do
not see the individual grass plants.

f)

Counting Bacteria using
Dilutions



There are times you need to know
how many bacteria you have in a liquid
culture. Since there may be 10
9

bacteria per milliliter
-

1,000,000,000


it may be too hard to count
them directly under a microscope…. So an easy way to count them is to
dilute the original culture; count the bacteria in the dilution, and use
some basic
math to calculate how many were in the original culture.
There are two basic ways to do this
-

plate out a sample of the
dilution by spreading on plates and waiting for colonies to form and
count the colonies, or count the diluted bacteria directly using a

hemocytometer

g)


Making
serial dilutions

i)

http://abacus.bates.edu/~ganderso/biology/resources/dilutions.ht
ml


ii)

To be able to count colonies on a plate, there should be
(statis
tically) between 30 and 300 colonies, fewer than 30 and a
randomness is not assured


greater than 300 (250 is actually
better), it is difficult to count the colonies accurately

iii)


A dilution is where a small sample of bacteria is placed in a sterile
test tu
be, mixed with a liquid (usually nutrient broth or saline) so
the bacteria are evenly distributed throughout the tube, and then
a sample is used for counting.

iv)

Because they are SO many bacteria in the original sample, very
often we need to dilute the diluti
ons to get a number of bacteria
we can count on a plate. This dilution series is called serial
dilutions.

4)

Activities

a)

Using a Microscope (oil lens)

i)

http://www.udel.edu/scope


ii)


Any basic microscope lab PLUS how to u
se oil lens

iii)

Emphasize to use ONLY lens paper to clean lens


regular tissues
or Q
-
tips can scratch lenses (scratch the coating on the lens)

iv)

Be sure to teach how to clean immersion oil off lens and need to do
so to prevent damage since old immersion oil wil
l crystallize on lens
causing damage
.
U
se naphtha, xylene, or turpentine (use very small
amounts on lens tissue)

as solvent
.

Do not use water, alcohol or
acetone, as the oil is insoluble to these solvents.

v)

http://docs.google.com/viewer?a=v
&q=cache:kI0dJ7JYWn8J:nic.m
ed.harvard.edu/equipment/help_sheets/Care%2520and%2520Clea
ning%2520of%2520your%2520Microscope.pdf+microscope+cleanin
g+procedure+oil+lens&hl=en&gl=us&pid=bl&srcid=ADGEESgN2ehlCC
D8RZyoGxJPInlLtxAujVURrMIFl8Xo9Ortd_LQAqFyPxl5gTrMzW
D
vDDDRXOmwm2GZLPIFJJ6Phvg5tnjJRYhtBYpnh8ftrO00IAXIH
m3BGlMkvYn6I0cpZyocDYjQ&sig=AHIEtbQAmZBpR3gdXFyh5u
-
UwFmaJ8aiNg


vi)

Lab 2C, 2d


Daughtery Lab Book

b)

Pipetting and Micropipetting practice

i)

Lab 3a, 3b, 3d in Daughtery

c)

Using a scale/balance

i)

Lab 3c daughtery

d)

Solu
tion making

i)

Lab 3e, 3f, 3g, 3h Daughtery

e)

Making Media

i)

Lab 4e, 4f Daughery

f)

Growing overnight cultures

i)

Lab 4G Daughery

g)

Streak for isolation

i)

Lab 4G Daughery

h)

Plating out

i)

Lab 4G Daughery