Notes from July 31

Bacterial Collection Techniques

 A microbiological culture, or microbial culture, is a method of multiplying microbial

organisms by letting them reproduce in predetermined culture media under
controlled laboratory conditions. 
 Microbial cultures are used to determine the type of organism.
 Bacteria is a microorganism which ranges in size from 0.5 – 1.0 µm
 Can be observed by light microscopy
 Bacterial samples are collected using Aseptic Technique
 In most clinics and laboratories, bacterial samples are collected with a swab
 After the swab sample is collected, bacteria must be grown on a special plate called
the agar plate

Transport Media

There are two (2) types of transport media:

1. Universal Transport Media
 Also known as AMIES
 Universal Transportation Medium (UTM) is room temperature, sterile, stable medium
which can sustain viability (and infectivity) of microorganisms, chlamydia,
mycoplasmas, or ureaplasmas during transit to the testing laboratory. 

2. Charcoal Transport Media

 Charcoal neutralizes fatty acids that are toxic to microorganisms
 Used in order to protect specimen from light and to maintain appropriate pH level
 Charcoal is a non-nutritional, phosphate buffered type medium used to maintain the
viability of microorganisms without a significant increase in growth
 It is important that relative numbers of pathogens and normal flora don’t change
Stool preservatives for C&S contains Na Thioglycolate (Cary Blair)

Charcoal
UTM:
Culture Media (Agar Plates)

The three (3) basic forms of culture media

1. Media poured and set in a semisolid form in Petrie dishes or plates.  These are
streaked with a technique that will spread out or dilute the culture to yield isolated
colonies for study and transfer. The plates are incubated in an inverted position and
examined at 24 and 48 hours

2. Slant media is poured into glass tubes that are covered and set at an angle. This
form of culture media can be used for maintenance as stock or reference cultures as
well as to culture certain organisms such as tubercolosis bacteria, which needs an
enclosed and secure system.

3. Liquid media, used to grow and confirm organisms by color changes (broth). Bacteria
are free to move around. Excretory products of bacterial metabolism pass into the
media.
A Petri dish (or Petri plate or cell culture dish) is a shallow glass or plastic cylindrical
lidded dish that biologists use to culture cells, plastic

 Petri dishes must be disposed after one use.

 Modern Petri dishes often have rings on the lids and bases which allow them to be
stacked so that they do not slide off one another.
 For microbiology, agar plates are very frequently used. The dish is partially filled with
warm liquid agar along with a particular mix of nutrients, blood, salts, sugars, dyes,
indicators, amino acids and/or antibiotics. After the agar solidifies, the dish is ready
to receive a microbial sample. 

 Petri plates are incubated upside down (agar on top) to keep the weight in the lid for
sterility, and so excess water accumulates away from the bacterial colonies.

To generate pure cultures from a mixed culture, use a technique known as "streaking or
Inoculation". In this technique, a drop of the culture on the end of a thin, sterile wire loop
or inoculator, is streaked across the surface of the agar leaving organisms behind, a
higher number at the beginning of the streak and a lower number at the end, this is
called “the Quadrant Technique” 

 Agar plates may be formulated as either permissive, with the intent of allowing the
growth of whatever organisms are present, or restrictive or selective, with the intent of
only allowing growth of a particular set of those organisms

Classification of Culture Media

The culturing material used is in order to grow bacteria present in the body fluid.

Media must have adequate moisture, carbon dioxide and protein nitrogen. The addition
of blood, vitamins and inorganic salts enhances the culture of certain bacteria. Other
factors influencing the growth of bacteria are the presence or absence of oxygen, pH
and temperature.

Also, media must be sterilized at temperatures that preserve the nutritional properties,
but destroy any organisms present. This ensures that the organism that is inoculated is
the one which is grown.
CDC estimates Salmonella bacteria cause about 1.35 million infections, 26,500
hospitalizations, and 420 deaths in the United States every year.

The pH of the culture media is usually neutral (7.0) Empty Petrie dishes must be
sterilized by ethylene oxide gas done commercially No bubble should be present on the
plate.

Selective Media

 Selective media promotes the growth of one type of organism while inhibiting the
growth of others
 Only select microorganisms can grow on it
 Antiboitics, dyes, or chemicals can be added that inhibit certain organisms
 Example: MacConkey, it is selective as it has crystal violet and bile salts that inhibit
the growth of gram positive bacteria (It is used to recover gram negative Bacteria). 
Differential Media

 Organisms are grown with specific nutrients or indicators added to the media to
distinguish one organism from another as seen by color change
 Example: MacConkey, E.coli as a gram negative, it grows and ferments lactose by
forming acid product that changes the PH of the media and it appears pink in color. 
 Salmonella and Shigella (stool Pathogens) do not ferment lactose and appear
colorless.

Supportive Media

 It contains nutrients that permit the growth of non-fastidious organisms. 

coli (Escherichia coli), is a type of bacteria that normally lives in your intestines. It's also
found in the gut of some animals. Most types of E. coli are harmless and even help
keep your digestive tract healthy. But some strains can cause diarrhea if you eat
contaminated food or drink fouled water.

Shigella infection (shigellosis) is an intestinal disease caused by a family of bacteria

known as shigella. The main sign of shigella infection is diarrhea, which often is
bloody. Shigella can be passed through direct contact with the bacteria in the stool.
People can get shigellosis when they put something in their mouths or swallow
something that has come into contact with the stool of someone else who is sick
with shigellosis. People could get sick by: Getting Shigella germs on their hands and
then touching your food or mouth.

 
Symptoms of shigellosis include:

 Diarrhea (sometimes bloody)
 Fever.
 Stomach pain.
 Feeling the need to pass stool [poop] even when the bowels are empty.

salads (potato, tuna, shrimp, macaroni, and chicken), raw vegetables, milk and dairy
products, and poultry can carry Shigella bacteria. Water contaminated with human
waste and unsanitary handling by food handlers are the most common causes of
contamination in these food products.

Supportive Media
 It contains nutrients that permit the growth of non-fastidious organisms. 

FACTORS INVOLVED IN MEDIA PREPARATION  

A. Filtering heat sensitive ingredients used in media making is not an easy proposition.
There are a number of types of filters available. The most satisfactory filter is the Seitz
filter. This consists of a metal unit with a series of filters of varying pore size. The
equipment is attached to a vacuum pump that draws the ingredients through to a
collecting flask. The Seitz filter is capable of filtering out bacteria if the correct sized
pore filter is used.

There are other filters available as well:

1. Berkefeld filter, composed of diatomaceous earth impermeable to ordinary bacteria,

i.e., bacteria cannot go through the filter.
2. Membrane filter, made up of a thin film of collodian, cellulose acetate or other
material. A wide range of pore sizes are available the smaller ones are able to retain
some viruses.
3.  Millipore filter. A trade name for a filter that, it is stated will filter out viruses.

The measurement of pH has been mentioned previously. The pH value may be

determined by an indicator system or by a pH meter. The latter is the method of choice
since it measures the hydrogen ion concentration directly. A pH level can be determined
now by using a flat electrode on the surface of the medium. It is sometimes useful to
use this type of electrode system on media such as MullerHinton for sensitivity testing.

Inspissation. The term inspissate means to be thickened, dried or made less fluid by
evaporation.

However, we use the term for the treatment of media following manufacture and tubing.
Tubes of media are placed on a rack and placed into the instrument. The instrument is
set to heat up the medium to a temperature of approximately 70oC, i.e., so that the
medium will not boil. Media are left at this temperature for one to two hours. The
process is usually repeated on three successive days. The coagulation of materials
such as serum or egg albumen occurs during the inspissation (Serum is used in
Loeffler's medium, eggs in Lowenstein-Jensen medium for MTB).
Mueller-Hinton agar is a microbiological growth medium that is commonly used for
antibiotic susceptibility testing. It is also used to isolate and maintain Neisseria and
Moraxella species.
Neisseria is a large genus of bacteria that colonize the mucosal surfaces of many
animals. Of the 11 species that colonize humans, only two are pathogens, N.
meningitidis and N. gonorrhoeae.
A number of common childhood illnesses, including some middle ear (otitis media) and
sinus infections (sinusitis), are caused by Moraxella catarrhalis bacteria. On rare
occasions, this same organism may cause a blood infection (bacteremia), an
eye infection (conjunctivitis), and meningitis in newborns.
Inspissation. The term inspissate means to be thickened, dried or made less fluid by
evaporation. However, we use the term for the treatment of media following
manufacture and tubing. Tubes of media are placed on a rack and placed into the
instrument.
The instrument is set to heat up the medium to a temperature of approximately 70oC,
i.e., so that the medium will not boil. Media are left at this temperature for one to two
hours. The process is usually repeated on three successive days. The coagulation of
materials such as serum or egg albumen occurs during the inspissation (Serum is used
in Loeffler's medium, eggs in Lowenstein-Jensen medium for MTB).

Aseptic dispensing must be used for all types of media.

This is accomplished by having all sterile glassware for manufacture as well as (as far
as possible) sterile solvent and other materials. Following manufacture one must
sterilize the prepared medium. The dispensing is performed (such as pouring the
medium into sterile plates) in an area devoid, if possible, of air currents, etc. The flask
containing the media should be flamed around the neck to avoid any possible
contamination. If plates are being poured the surface of the medium on each plate
should have a Bunsen flame quickly passed over the surface of the medium to dissipate
bubbles if present, but also to coagulate any bacteria that may be on the surface of the
plates. For a number of years now an automatic media dispenser has been available.
Most labs today purchase ready-made culture media from a supplier who specializes in
this field.

Sterilization is the complete removal or destruction of all living organisms including

bacteria and spores*. There are several means by which sterilization may be
accomplished.

1. Dry heat - temperatures of about 160oC for 1 hour are necessary to kill spores. This
method is used mainly on glassware, metal instruments and certain fatty substances
that are not permeable to water.

2. Wet heat - (steam under pressure) - The most efficient method of sterilization.
Requires heat at 121oC for 15-30 minutes. Moist heat accomplishes sterilization by
denaturing the enzymes and proteins of micro-organisms.

Three methods used

1. Moist Heat:

a. Moist heat and temperature below 100oC, e.g., mild pasteurization or inspissation of
media (will not kill spores).

b. Moist heat and temperatures at 100oC. Boiling and steaming are typical examples.
The Arnold Steam Sterilizer is one example. Another is the boiling water sterilizer used
for instruments. (Disinfects only - does not kill spores).
c. Moist heat and temperatures above 100oC. The autoclave (steam under pressure) is
the most reliable method. The steam is allowed to permeate all articles for sterilization
at a pressure of 15 lbs. per square inch. This attains a temperature of 121oC.

2. Radiation Method. Ultraviolet rays with shorter wavelengths are effective in killing
bacteria. Mercury vapour lamps with wavelengths of 240-280 nm are most effective.
This method is used to sterilize rooms or the seeding cabinet in the lab. Beware of UV
light; one can receive burns to eyes by looking directly at the UV lamp.

3. Filtration. As previously mentioned, one can use a Seitz or Berkefeld filter system.
Cellulose membrane filters are most effective and can be used to retain bacteria for
culture.

1. Chemical Disinfection. Disinfectants can only be used for certain purposes, e.g.,
swabbing down benches to prevent infection or spread of infection. Phenol has been
used as the typical example of this method and all other disinfectants are compared
with it. The ideal bactericidal and virocidal agent is a dilute hypochlorite solution
(Javex/Chlorox 1-2 %)

MEDIA ADDITIVES

1. Defibrinated blood - Sterile flask with sterile glass beads (3 mm in diameter). Collect
blood with a sterile syringe using aseptic technique and eject into the flask. Shake
the blood from side to side for 10 minutes. The fibrin formed during clotting will be
deposited on the beads. Decant the supernatant containing blood cells and serum
into a sterile container and store in the refrigerator. Label with contents and date of
preparation.
2. Lake blood. Laking of blood is the separation of hemoglobin from the red cells. This
can be performed in a number of ways. A simple method is to add cold sterile
distilled water to the blood in a sterile container. Centrifugation, will deposit the red
cell stroma at the bottom of the tube flask. Hemoglobin is required for a number of
types of media particularly for growth of Neisseria. One can purchase a pure
hemoglobin powder for reconstitution with distilled water. This serves as a substitute
for laked blood.
3. Eggs in media - typical examples of the use of eggs in media are the requirements in
both Lowenstein-Jensen medium and Petragnani medium. In both instances, a large
volume   of eggs is required and these are wiped with an alcohol sponge and
allowed to dry. They are cracked into a sterile container using sterile instruments
and using an aseptic technique entirely. Great care has to be taken not to
contaminate the eggs. In the case of both media, a sterile solution containing
 necessary ingredients is transferred to the container of eggs. This is mixed well and
a quantity of malachite green is added to prevent contamination. Inspissation is used
after placing the media in tubes.
4. Carbohydrate solutions. These are 20% solutions of glucose, maltose, sucrose, and
lactose. They are added individually to a basal fermentation medium for fermentation
studies of aerobes and anaerobes for identification purposes.
5. Buffers are used extensively in media making. Each individual medium contains
these. The usual buffers are phosphate buffers (sodium and potassium phosphates
in the monobasic and dibasic state.

Common Types of Agar Plates/Media Types of Blood Agar (Universal Media)

Blood agar plate (BAP) 

 Contains mammalian blood (usually sheep), typically at a concentration of 5–10%. 

 BAP are enriched, differential media used to isolate fastidious organisms and detect
hemolytic activity. 

 Contains meat extract, tryptone, sodium chloride, and agar.

 Widely used to differentiate between Streptococcus species according to the action

of  hemolysis:

 β-hemolytic activity will show complete hemolysis seen as a clear zone surrounding
colony. Example include Streptococcus pyogenes.

 Group A strep can cause pharyngitis (strep throat). It can also cause
glomerulonephritis, rheumatic fever, scarlet fever, necrotizing fasciitis

 α-hemolysis is partial hemolysis seen as a green discoloration of the media

surrounding the colony 

 Example of this would be Streptococcus viridans, Strep. pneumonie.  

 γ-hemolysis (or non-hemolytic) is the term referring to a lack of hemolytic activity. 

 Scarlet fever is a bacterial illness that develops in some people who have strep
throat. Also known as scarlatina, scarlet fever features a bright red rash that covers
most of the body. Scarlet fever is almost always accompanied by a sore throat and a
high fever.
ecrotizing fasciitis (NF), also known as flesh-eating disease, is an infection that results
in the death of parts of the body's soft tissue. It is a severe disease of sudden onset that
spreads rapidly. Symptoms usually include red or purple skin in the affected area, severe
pain, fever, and vomiting.
Other names: Flesh-eating bacteria, flesh-eating ...
Treatment: Surgery to remove the infected tiss...
Symptoms: Severe pain, fever, purple colored
Necrotizing fasciitis (NF), also known as flesh-eating disease, is an infection that results
in the death of parts of the body's soft tissue. It is a severe disease of sudden onset that
spreads rapidly. Symptoms usually include red or purple skin in the affected area, severe
pain, fever, and vomiting.
Other names: Flesh-eating bacteria, flesh-eating ...
Treatment: Surgery to remove the infected tiss...
Symptoms: Severe pain, fever, purple colored
This group of Streptococci are most often found in the mouth, gut and genital region.
The most serious Viridans infections occur when the bacteria enters other regions of
the body. For example, if Viridans gets into the bloodstream it can
cause endocarditis (infection of the inner lining of the heart).
Streptococcus pneumoniae is a bacterium that causes respiratory infections in children
and adults as well as meningitis. It is the most common cause of bacterial inner ear
infection in children. It is common in adults and the most frequent cause
of pneumonia among the elderly and those not able to fight off infections.

Chocolate agar (CHOC) 

 A type of blood agar plate in which the blood cells have been lysed by heating the
cells to 56 °C. (No chocolate is actually contained in the plate; it is named for the
coloration only).
 Chocolate agar is used for growing fastidious (fussy) bacteria, such as Haemophilus
influenzae. 
 CSF cultures plated on chocolate
A fastidious organism is any organism that has complex or particular nutritional
requirements. ... The more restrictive term fastidious microorganism is used in
microbiology to describe microorganisms that will grow only if special nutrients are
present in their culture medium.

Haemophilus influenzae (formerly called Pfeiffer's bacillus or Bacillus influenzae) is a

Gram-negative, coccobacillary, facultatively anaerobic capnophilic pathogenic bacterium
of the family Pasteurellaceae. H. influenzae was first described in 1892 by Richard
Pfeiffer during an influenza pandemic.

Thayer-Martin agar (TM) 

 Chocolate agar designed to isolate Neisseria gonorrhoeae. It is made selective by

adding antibiotics to inhibit normal flora and fungus.

 Also used for Neisseria meningitidis

TCBS agar 

 TCBS (Thiosulfate Citrate Bile Salts Sucrose) - enriched agar enhances growth of
Vibrio spp., including Vibrio cholerae

Mannitol salt agar (MSA) 

 MSA is also a selective and differential media. The Mannitol indicates organisms
that ferment Mannitol, mannitol fermentation produces lactic acid, lowering the pH
and turning the plate yellow. 
 The salt is to select for halophiles; organisms that cannot withstand a high salt
content will be unable to grow well. 

Mueller-Hinton agar (MHA) : MHA contains beef infusion, peptone, and starch and is
used primarily for antibiotic susceptibility testing. It can be in a form of blood agar. 
Mueller-Hinton has a few properties that make it excellent for antibiotic use. ... Starch
is known to absorb toxins released from bacteria, so that they cannot interfere with the
antibiotics. Second, it is a loose agar. This allows for better diffusion of the antibiotics
than most other plates.
Nutrient agar 

 Nutrient agar is usually used for growth of non-fastidious organisms and observation
of pigment production. 

 It is safe to use in school science laboratories because it does not selectively grow
pathogenic bacteria. 

Önöz agar/ also called (SSA: Salmonella Shigella Agar): Önöz agar allows more rapid
bacteriological diagnosis as Salmonella and Shigella colonies which can be clearly and
reliably differentiated from other Enterobacteriaceae. 

Phenylethyl alcohol agar (PEA) : PEA selects for Staphylococcus species while
inhibiting Gram-negative bacilli (e.g. Escherichia coli, Shigella, Proteus, etc.). 

R2A Agar (R2A) : Used for water analysis. 

Tryptic (Trypticase) Soy Agar (TSA) 

TSA plates support growth of many semi-fastidious bacteria, including some species of
Brucella, Corynebacterium, Listeria, Neisseria, and Vibrio. 

Xylose-Lysine-Deoxycholate agar (XLD) 

 Cetrimide agar 

Cetrimide agar is a type of agar used for the selective isolation of the gram-negative
bacteria, Pseudomonas aeruginosa. 

SMAC

Sorbital MacKoncey is a selective and differential media for E.collie 0157 H7 which
causes hemorrhagic colitis

 XLD is used for the culture of stool samples and contains two indicators. It is
formulated to inhibit Gram-positive bacteria, while the growth of Gram-negative
bacilli is encouraged. 

 The colonies of lactose fermenters appear yellow. 

 It is also used to culture possible Salmonella that may be present in a food sample. 

 Salmonella colonies will show a black halo on XLD.

Brucellosis is an infectious disease caused by a type of bacteria called Brucella. The
bacteria can spread from animals to humans. There are several different strains
of Brucella bacteria. Some types are seen in cows. Others occur in dogs, pigs, sheep,
goats, and camels

Pseudomonas aeruginosa is a common encapsulated, Gram-negative, rod-shaped

bacterium that can cause disease in plants and animals, including humans.