Download Lecture 07 - Laboratory Procedures

Laboratory Procedures
Bacteria
Culturing Bacteria
Culturing: To introduce a specific
microorganism into a solid or liquid broth
in order to grow that microorganism for
laboratory testing.
 Four steps:





Swabbing the surface/patient you want to
culture.
Streaking the swab onto an agar plate.
Staining/testing for identification.
Maintaining a stock culture.
Culturing A Patient

Proper Method:



Place cotton swab against
area being cultured.
Gently spin the swab or
move side-to-side to
maximize number of
microorganisms picked
up.
Lift swab away from
surface and then remove
from area.
Streaking Onto Agar

The first step we must take is to learn
about agar.
Agar

Agar: A complex polysaccharide medium
used to grow microorganisms.


First used by Koch when developing his
postulates, but still used to this day!
Begins as a solid, but can be melted into a
liquid.


Once melted, it does not solidify until it cools to
40˚C/104˚F.
Allows heat-sensitive substances to be added just
before solidification.
Agar

Basic agar is made
by combining either
chicken or beef broth
with a base agar
powder.


Some types of agar
go father than this!
Peptone: A product
of enzyme digestion
of proteins easily
utilized by
microorganisms – a
common ingredient!
Agar

Bacteria divides
exponentially on
either solid or liquid
agar.


Liquid agar provides a
mixed culture
populated by several
bacteria.
Solid agar provides
growth in colonies that
are decedents of a
single cell.
Streaking Onto Agar

The second step is to figure out which
type of agar you want to use, based on
patient symptoms.
Classes of Agar
Synthetic Media: A medium prepared in
the laboratory from materials with a
precise or reasonably well-defined
composition.
 Complex Media: A medium with a similar
base composition as synthetic media, but
with slightly differing composition from
batch to batch.


E.g.: Could contain beef, yeast or soy protein,
in any combination.
Classes of Agar

Selective Medium: Encourages the
growth of some bacteria but discourages
the growth of others.


Typically involves including certain antibiotics
that will not harm the particular bacterium it is
designed to grow.
Used to try to grow just one type of organism.
Classes of Agar

Differential Medium: Contains a
constituent that causes an observable
change.



Typically has chemicals that will induce a
chemical reaction and color change when
certain organisms are present.
This lets us tell different colonies apart based
on color change.
Used to tell things apart!
Classes of Agar

Enrichment Medium: Contains special
ingredients to allow a particular organism
to flourish.


Especially useful when there may not be
enough organisms to be isolated and identified
using a normal agar method.
Problem: It does not suppress the growth
of other organisms – EVERYTHING goes
wild!
Types of Agar –
Blood Agar

Defining Feature:


Contains 5% blood cells
from an animal, usually
sheep.
What Will Grow:

Most bacteria will grow
on this.
Types of Agar –
Chocolate Agar

Defining Features:



Made of lysed red blood
cells, typically sheep.
Nutrient medium used to
culture fastidious
organisms.
DOES NOT give
aemolysis data
Types of Agar –
Chocolate Agar

What Will Grow:
Most bacteria will grow
on this.


Haemophilus grows due
to X & V factors from
the sheep blood –
cannot differentiate
between the species
though!
Neisseria grows due to
nutrient-rich
environment.
Types of Agar –
Luria Bertani (LB) Agar

Defining Feature:



A subtype of nutrient agar.
General medium used for
routine cultivation.
What Will Grow:



Does not grow fastidious
microorganisms.
Does not preferentially grow
one kind of bacteria over
another.
Grows everything!
Types of Agar –
Miller’s LB Agar

Defining Feature:



A variation of LB agar.
Contains the same basic
components of LB agar, but
in different proportions.
What Will Grow:

Only Gram Negative
bacteria will grow.
Types of Agar –
MacConkey Agar

Defining Feature:



E. coli differentiates by
growing into red colonies
due to sugar fermentation.
Can be mixed with or
without sugar lactose.
What Will Grow:

Will only grow Gram
Negative bacteria.
Types of Agar –
Neomycin Agar

Defining Feature:



Contains neomycin (antibiotic).
Used to culture anaerobic
microorganisms.
What Will Grow:



Only species not sensitive to
neomycin will grow.
Stops the growth of Gram
Negative bacilli and
staphylococci.
Allows streptococcus species to
grow abundantly.
Types of Agar –
Neomycin Agar

Interesting Fact: Neomycin was
discovered in 1949 by the microbiologist
Selman Waksman. P



Produced naturally by the bacterium
Streptomyces fradiae.
Has a broad spectrum of effects, killing both
gram-positive and gram negative bacteria.
Relatively toxic to humans, and some people
have allergic reactions to it.
Types of Agar –
Non-Nutrient Agar

Defining Feature:


May be used to grow nonbacterium
microorganisms.
What Will Grow:


Does not grow bacteria.
May be used for growing
other microorganisms.
Types of Agar –
Trypticase Soy Agar (TSA)

Defining Feature:




Grows the widest variety of
microbes.
Uses beef broth.
Contains some yeast
extracts.
What Will Grow:


Will grow bacteria & fungi.
Doesn’t grow all bacterium –
some find it too rich, others
find it deficient.
Types of Agar –
Sabouraud Agar

Defining Feature:




Used to grow fungi.
Has a low pH (kills most
bacteria).
Contains gentamicin antibiotic
(particularly useful for Gram
Negative bacteria).
What Will Grow:

Grows fungi.
Types of Agar –
Thayer-Martin Agar

Defining Feature:


Designed to isolate Neisseria
gonorrhoeae.
What Will Grow:

Specifically designed for
Neisseria gonorrhoeae.
Types of Agar –
Tryptic Soy Agar (TSA)

Defining Feature:


A basic medium for
general culturing.
Mainly used as an initial
growth medium for…





Observing colony
morphology.
Developing pure cultures.
Gaining sufficient growth for
biochemical testing.
Culture storage.
What Will Grow:

Just about everything!
Types of Agar –
Xylose Lysine Deoxychocolate Agar

Defining Feature:




Used for culturing stool samples.
Contains two different
indicators.
Colonies of organism that
ferment lactose will appear
yellow.
What Will Grow:


Formulated to inhibit Grampositive bacteria.
Formulated to encourage Gramnegative bacilli.
Streaking
Streaking: The specific technique for
isolating bacterium on solid agar.
 Streak Plate Method: A method used to
obtain colonies from a single parent
species.

Streak Plate Method
1. The streaking tool is
held in open flame to
sterilize, then dipped
into a colony or culture.

In the case of a patient
culture, it is applied
directly to the agar.
2. The culture is moved in
a back-and-forth motion
across the top of the
plate.
Streak Plate Method
3. The plate is turned 90˚
and a sterile loop is used
to streak down from one
corner, only touching the
previously streaked
region once, then
continuing up and down
the far side of the plate.
4. The plate is turned 90˚
again, and a sterile loop
is used to repeat the
streaking process.
Staining For Identification

Staining serves several purposes…



Tells us information about the cell wall.
Helps us to identify the microorganism.
Helps us to figure out what chemotherapeutic
agents will kill it.
Types of Stains

Stains and dyes are molecules that bind
to a cellular structure to give it color.

Cationic Dyes: Basic, positively charged dyes
that are attracted to negatively charged cell
components.


Includes methylene blue, crystal violet, safranin, &
melachite green.
Anionic Dyes: Negatively charged dyes that
are attracted to positively charged material.

Includes eosin & pictric acid.
Methods of Staining

Simple Stain: Uses a single dye.



Allows us to determine cell shape and
arrangement under a microscope.
Can be done with methylene blue, safranin,
crystal violet, and carbolfuchsin.
Differential Stain: Uses two or more
dyes.


Allows us to distinguish between the different
parts of an organism and between organisms
close to each other.
Examples: Gram Stains and Ziel-Neelsen
Acid-Fast Stain.
Gram Stain

Gram Stain: A staining method that
allows us to determine the type of cell wall
a bacterium possesses.






The bacteria are streaked onto a glass slide.
The slide is passed over a flame to heat-fix the
material.
The slide is flooded with crystal violet (purple)
for 15 seconds.
The slide is rinsed with iodine, then water.
The slide is flooded with safranin (red) for 15
seconds.
The slide is light rinsed with water and pat dry.
Gram Stain
Gram Stain

Reminder:




Gram-Positive & Acid-Fast bacteria retain
the crystal violet due to the thickness of
their cell walls.
Gram-Negative bacteria loose the crystal
violet and keep the safrinin (since no
alcohol rinse is used to remove it!) due to
the thinness of their cell wall.
Gram-Variable cells stain purple and red –
usually due to the cells being too old when
cultured, which decreases their ability to
retain stains.
This is a differential stain.
Gram Stain
Ziehl-Neelsen Acid-Fast Stain

Ziehl-Neelsen Acid-Fast Stain: Tests
for the high lipid content of their cell walls.

The same basic procedures are used as in
Gram Staining, except…


Crystal violet is replaced with carbolfuchsin (a red
stain).
Safranin is replaced with a blue dye.
Acid-Fast bacteria stains red from the
carbolfuchsin.
 Bacteria that stains blue should be Gram
stained.
 This is a differential stain.

Ziehl-Neelsen Acid-Fast Stain

Tuberculosis:
Flagellar Stain
Used to identify specific
structures within an
organism.
 Specifically, flagellated
bacteria appear dark with
flagella appearing as silver
or red lines.
 This is a special stain.

Stock Culture

Pure Culture: A culture containing only
one species of organism.


Stock Culture: A culture grown in liquid
broth from one organism or one colony of
organisms.


Streak Plate Method is used to obtain singlespecies colonies.
This can be innoculated onto fresh agar when
the bacteria is needed for study.
Aseptic Techniques and other measures
must be used to prevent contamination of
the stock culture.
Measuring Bacterial Growth

Turbidity: The cloudy
appearance of a liquid
broth with containing
bacterial colonies.
Measuring Bacterial Growth

Spechtophotometer: An
instrument used to measure
the degree of light
transmission through a
turbid culture.



Allows an indirect
measurement of the number
of cells present in the culture.
Allows measurement without
disturbing the culture.
Can be inaccurate if there are
too many or not enough
Measuring Bacterial Growth

Standard Plate Count: Estimates the
number of bacteria from a culture
assuming that each colony grown on a
solid agar plate originated from a single
organism.
Measuring Bacterial Growth

Serial Dilutions: A method of diluting a
liquid culture to prevent over-crowding of
colonies when grown on solid agar.


Multiplying the number of colonies by the ratio
of dilution to estimate the number of bacterial
cells in the original culture.
For example: If a microbiologists dilutes a
broth to 1:1000 ratio of broth to dilutant, and
50 colonies grow on agar by the next day, he
can estimate the 50,000 bacterial cells per one
milliliter of original culture.
Measuring Bacterial Growth

Most Probable Number (MPN): An
estimation of the number of bacteria that
is used when there are too few cells to use
other methods.
Measuring Bacterial Growth

Most Probable Number (MPN):




Measures the bacterial waste byproducts
produced.
A small glass tube is inverted & placed into the
test tube containing liquid culture so that it fills
with broth.
Gas produced by the fermentation of bacteria
will be trapped in the inverted tube.
The gas can then be compared to a
standardized curve to estimate the number of
organisms present in the sample.
Filtration

Filtration: Forces a known volume of
liquid or air through a filter with pores too
small for bacteria to pass through.



The filter can then be placed on solid media
and allowed to grow.
The resulting colonies can then be counted
using standard plate count.
The standard plate count allows an estimate of
the number of organisms per liter of air or
water to be estimated.
Fungi
Culturing Fungi
Fungi can be grown, cultured and
identified using similar laboratory methods
to those used on bacteria!
 Acidic, high-sugar media with
antibiotics to prevent bacterial growth can
be used.
 Most pathogenic fungi take two to four
weeks to grow as much as bacteria do in a
24-hour period.
 This leads to slow identification!

Types of Agar –
Sabouraud Agar

Defining Feature:




Used to grow fungi.
Has a low pH (kills most
bacteria).
Contains gentamicin
antibiotic (particularly
useful for Gram Negative
bacteria).
What Will Grow:

Grows fungi.
Viruses
Culturing Viruses
Whole animals had to be used to culture
viruses until the 1950’s and 1960’s.
 Two big discoveries changed this…



The discovery of antibiotics helped with
culturing viruses and preventing bacterial
contamination of the culture.
The discovery of proteolytic enzymes,
especially trypsin, that helps to free animal
cells from their surrounding tissue without cell
injury – this allowed us to add cells to Petri
dishes to grow viruses in!
Culturing Viruses

Primary Cell
Cultures: Cultures
that come directly
from an animal.



The younger the source
animal, the longer the
cell will survive within
the culture.
Typically presents as a
mixture of cell types.
Only divide a few times,
but can host a wide
variety of viruses.
Culturing Viruses

Continuous Cell Lines: Cells that will
reproduce for an extended number of
generations, allowing long-term study.

Typically originate from cancerous cells or cells
that have lost control of cell division rates.
HeLa Cells

HeLa Cells: An
immortal cell line used in
medical research,
particularly with
virology.

HeLa Cells





Started from a cervical cancer
cell culture taken from Henrietta
Lacks.
Mrs. Lacks died on October 4th,
1951.
Her cells are located in just about
every laboratory in the U.S.
These cells can divide indefinitely
as long as basic conditions for
survival are met.
They have branched into several
strains as they have mutated
over the years.