Exercise 9 (page 87, table 9.

1)
• B. cereus is a sporeformer
– Can withstand most chemicals
– Should see growth, but it may decrease over time
• E. coli
– Most Gram – bacteria are susceptible due to outer membrane
– Shouldn’t see too much growth
• S. aureus
– Intermediate
– Should see some growth (less than B. cereus but more than E.
coli)
Exercise 10 (page 93, table 10.1)

• Observe differences between the 2 sides of the plate

• Which method reduces the amount of growth &
why?
– Scrub and Sanitizer should have least amount of
growth
– Soap & Water may have some growth
– Water should have most growth
• Where there any anomalies?
– Can occur due to paper towels 
Exercise 11: Antibiotic Susceptibility Objectives
• Define antibiotic
• Differentiate between broad-spectrum & narrow-
spectrum antibiotics
• List 2 factors contributing to the development of
antibiotic-resistant strains of bacteria
• List 2 types of mechanisms of action of antibiotics
• Explain the importance of testing antibiotic
susceptibility
• Define & interpret zone of inhibition
• Explain why it is important to start with a pure culture
when performing an antibiotic susceptibility test
Antibiotics

• Penicillin was 1st (discovered by Fleming in 1929)

• Antibiotics are chemical compounds that selectively
inhibit or kill microorganisms while causing little or
no damage to animal cells
– Can be introduced to human body to treat
bacterial infections with minimal side effects on
human cells
• Must be used properly to ensure their clinical
usefulness
Antibiotics

• Table 11.1 lists some common antibiotics, effects on cells,

& spectrum activity
• Some affect cell wall production of bacteria, inhibit
protein synthesis in bacterial cell, & others disrupt cell
membranes
• Broad-spectrum antibiotics are effective against a wide
variety of bacteria & affects an aspect of the cell such as
protein synthesis
• Narrow-spectrum antibiotics are effective against specific
bacteria targeting either Gram + or Gram – bacteria
Resistance
• Resistance Mechanisms:
– Inactivation of the drug by the bacterium
– Blocking or mutation of the active site of drug action in the
bacterial cells
– Preventing the drug from entering the cell & reaching the
active site of drug action
• To become resistant bacteria often have to make a single
resistance protein
– Single genes required to synthesize such protein is encoded
on a plasmid
– Plasmids can be transferred between neighboring cells
 Testing
• Once bacterium is isolated from a patient,
the antibiotics needed must be determined
• Kirby-Bauer method is on of the oldest
methods to test
– Mueller-Hinton agar is inoculated with
pure culture
– Filter paper disks containing antibiotic are
placed on agar surface
– While plates are being incubated the
antibiotic diffuses through the medium
– If the antibiotic is effective, bacterial
growth around disk is inhibited (clear
zone)
– Diameter (in mm) of the zone of inhibition
is measured & compared to standard
Exercise 12: Transport of Specimens Objectives

• Define generation time

• List & understand the significance of the 3 specified
considerations regarding specimen collection
• Describe the purpose of the Bio-Bag system
• Explain how leaving a clinical specimen at room temp
will affect the diagnosis of infection
Applications

• Many microorganisms are

difficult to culture from patients
so collection procedures must
be followed to ensure its
survival
• As health care professionals you
will have to follow certain
guidelines on how to properly
collect and transport specimens
to the lab
Generation Time

• Generation time is the time it takes cells to double in

number
– For most pathogens this is 30 minutes
– Growth of bacteria on nutrient agar is rapid
– In body fluids bacteria also grow rapidly
Nurses

• The collection & delivery of patient specimens to the

lab fall under their responsibility
• Nurses must understand bacterial growth in order to
know how to collect it
3 Considerations for Collection & Transport

1. Microorganisms cultured from body fluid must

be determined to be pathogenic or normal
flora, so specimens need to be handled
properly to ensure results aiding in the proper
treatment
2. Use of proper sterile techniques to collect
specimens so correct therapy is provided to
patient
3. For fastidious bacteria special transport media
must be used to ensure their survival
Various Bacterial Transports

Urine & Sputum Anaerobic Liquid

Culture

Aerobic, Anaerobic, &

Mixed Culture Fecal
Procedure

• Thayer-Martin media is used to isolate N.

gonorrheoea
• We will not be using this bacteria or media, instead
we are using chocolate agar & N. lactamica (non-
pathogenic!)
– Results will be similar to those as using Thayer-
Martin & N. gonorrheoea
Exercise 13: Selective & Differential Media
Objectives
• Define and give an example of a selective media
• Define and give an example of a differential media
Applications

• Initial media used in a clinical setting is a selective

and/or differential media
• The media promotes isolate by inhibiting the growth
of normal flora
Background

• So far we have used general growth medium that

allows for multiple bacteria to grow
• A selective medium contains an added drug to
growth medium to obtain the bacteria of interest;
will inhibit the growth of bacteria such as normal
flora
• A differential medium contains a substance(s) that if
used by the organism will cause a visible change in
the organism
Examples
• Mannitol Salt Agar (MSA):
– Both a differential & selective medium
• Contains mannitol & a pH indicator that changes color
when the mannitol is fermented & acid is produced
• Salt concentration of MSA is 7.5%
– Of normal flora, only Staphylococcus species can grow (makes
it selective for Gram +)
• A color change from red to yellow indicates a pH
change because the organism can ferment mannitol
(makes it differential)
Examples

• Eosin Methylene Blue (EMB), Hektoen Enteric (HE), &

MacConkey:
– Inhibit growth of Gram + bacteria (makes them
selective for Gram - bacteria)
– Differentiate between lactose fermenting & non-
lactose fermenting (makes them differential)
• Most intestinal pathogens can’t ferment lactose so if a
stool sample is collected & sample shows fermentation
then it is known that it is not normal flora
Examples

• Blood Agar:
– ONLY a differential media
– But it does allow for distinguishing Streptococcus
& Staphylococcus based on their hemolytic
properties
Demonstration Plates: MSA

Media Organism Reaction Selective Differenti

al
Type Cultured Observed Property
Property
Mannitol
fermenta
MSA S. aureus Yellow Gram + tion/
Coagulas
e+

S. No Non-
MSA Gram + fermente
epidermis reaction
r
Demonstration
Plates: Blood Agar

Organism Reaction Selective Differential

Media Type
Cultured Observed Property Property

BA NOT Lysis
S. aureus RBC lysis
selective

NOT
BA S. epidermis No lysis Non-lysing
selective
Demonstration
Plates: EMB

Media Type Organism Reaction Selective Differential

Cultured Observed Property Property

EMB Metallic Lactose

E. Coli Gram - fermenter
green sheen

Non-lactose
EMB Salmonella No reaction Gram -
fermenter

EMB Klebsiella Brown Gram - Lactose

fermenter
mucoid
Demonstration
Plates:
HE/HEK

Organism Reaction Selective Differential

Media Type
Cultured Observed Property Property

HE Yellow- Lactose
E. Coli Gram - fermenter
Orange

Non-lactose
Black fermenter/prod
HE Salmonella centered Gram - uces hydrogen
colonies sulfide (H2S)

Non-lactose
HE Klebsiella No reaction Gram -
fermenter