Introduction to Microbiology

SCIMA2-B22

LO6

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 Recap: What was covered in LO2?
• Microbial Replication and growth
• Physical and Chemical growth requirements of microorganisms
• Classification of microbes based on their growth requirements
• Oxygen toxicity
• Biofilms formation
• Culture media used for microbial growth
• Obtaining pure cultures of microorganisms
• Preserving microbial cultures
• Reproduction in Prokaryotes
• The bacterial growth curve
• Calculating microbial growth
• Measurement of bacteria growth
Recap: Quiz
1. A medium with mannitol and high salt concentration can be
used for isolating. What type of media is this?
a. Staphylococcus
b. Candida
c. Escherichia
d. Thermovibrio
e. Campylobacter
 Recap: Quiz

2. Explain how each of the following terms is crucial to a

microbiologist:
a. BSL - 4
b. Coulter counters
c. Colony forming unit (CFU)
d. Lyophilization
 Recap: Quiz

3. A water sample is supposed to contain some coliforms and nitrifying

bacteria. Which direct method could help in the enumeration of the

bacteria?

4. Jennifer accidentally inoculated her chicken salad with five Salmonella

enterica cells. If S. enterica has a generation time of 30 minutes, how

many cells would be in her salad after 4 hours.

5. How are enrichment media different from selective media?

Resources for this lesson:

• Tortora et al. (Chapter 6)

• Lecture slides
• myLMS notes
- Including associated videos
Rate your Applied Science Lecturer
 Control of Microbial Growth

In this lesson we will look at the different

methods of microbial inactivation and the
control mechanism of these
methods. Microbes are inactivated using
various methods which depends on many
factors. These factors would be analyzed in
relation to different microbes. Mechanism of
inactivation of these methods would also be
analyzed.
 What will be covered
Week 4 in today’s lesson?
Lesson 7 Different methods used in microbial
inactivation and the control measures

LO6: Control of Mechanism of inactivation of control

measures and the factors contributing
Microbial Growth to the effectivity

Principles of effective disinfection.

Advantages and disadvantages of the

different groups of treatment
Q&A

• In what situation is
filtration the only
practical way to
eliminate undesirable
microbes?

10
The Terminology of Microbial Control

Learning Objective
• 7-1 Define the following key terms related to
microbial control: sterilization, disinfection,
antisepsis, degerming, sanitization, biocide,
germicide, bacteriostasis, and asepsis.

11
The Terminology of Microbial Control

• Sepsis refers to bacterial contamination

• Asepsis is the absence of significant contamination
• Aseptic surgery techniques prevent the microbial
contamination of wounds
The Terminology of Microbial Control

• Sterilization
• Removing and destroying all microbial life
• Commercial sterilization:
• Killing C. botulinum endospores from canned goods
• Disinfection:
• Removing pathogens / destroying harmful
microorganisms
• Antisepsis:
• Removing and destroying pathogens from living tissue

13
The Terminology of Microbial Control

• Degerming:
• the mechanical removal of microbes from a limited area
• Sanitization:
• lowering microbial counts on eating utensils to safe levels
• Biocide/germicide:
• Treatments that kill microbes
• Bacteriostasis:
• Inhibiting, not killing, microbes

14
Check Your Understanding

The usual definition of sterilization is the removal or

destruction of all forms of microbial life; how could there
be practical exceptions to this simple definition? 7-1

15
The Rate of Microbial Death

Learning Objective

• 7-2 Describe the patterns of microbial death caused

by treatments with microbial control agents.

16
Table 7.2 Microbial Exponential Death Rate: An Example
Figure 7.1a Understanding the Microbial Death Curve.
The Rate of Microbial Death

• Effectiveness of treatment depends on:

• Number of microbes
• Environment (organic matter, temperature, biofilms)
• Time of exposure
• Microbial characteristics
Figure 7.1b Understanding the Microbial Death Curve.
Check Your Understanding

How is it possible that a solution containing a million

bacteria would take longer to sterilize than one containing
a half-million bacteria? 7-2

21
Actions of Microbial Control Agents

Learning Objective

• 7-3 Describe the effects of microbial control agents

on cellular structures.

22
Actions of Microbial Control Agents

• Alteration of membrane permeability

• Damage to proteins
• Damage to nucleic acids

23
Check Your Understanding

Would a chemical microbial control agent that affects

plasma membranes affect humans? 7-3

24
Physical Methods of Microbial Control

Learning Objectives
• 7-4 Compare the effectiveness of moist heat
(boiling, autoclaving, pasteurization) and dry heat.
• 7-5 Describe how filtration, low temperatures, high
pressure, desiccation, and osmotic pressure
suppress microbial growth.
• 7-6 Explain how radiation kills cells.

25
Heat

• Heat denatures enzymes

• Thermal death point (TDP): lowest temperature at
which all cells in a liquid culture are killed in 10 min
• Thermal death time (TDT): minimal time for all bacteria
in a liquid culture to be killed at a particular temperature

26
Decimal Reduction Time (DRT)

• Minutes to kill 90% of a population at a given

temperature

27
A Microbial Death Curve

Figure 7.1b 28
Moist Heat Sterilization

• Moist heat denatures proteins

• Boiling
• Free-flowing steam
Moist Heat Sterilization

• Autoclave: steam under pressure

• 121 C at 15 psi for 15 min
• Kills all organisms and endospores
• Steam must contact the item's surface
An Autoclave

Figure 7.2 31
Moist Heat Sterilization

• Large containers require longer sterilization times

• Test strips are used to indicate sterility
Table 7.4 The Effect of Container Size on Autoclave Sterilization Times for Liquid Solutions*
Steam Sterilization

• Steam must contact item’s surface

Figure 7.3 34
Pasteurization

• Reduces spoilage organisms and pathogens

• Equivalent treatments
• 63°C for 30 min
• High-temperature short-time: 72°C for 15 sec
• Ultra-high-temperature: 140°C for 4 sec
• Thermoduric organisms survive

35
Dry Heat Sterilization

• Kills by oxidation
• Flaming
• Incineration
• Hot-air sterilization

Hot-Air Autoclave

Equivalent Treatments

36
Filtration

• Passage of substance
through a screen-like
material
• Used for heat-sensitive
materials
• High-efficiency particulate
air (HEPA) filters remove
microbes >0.3 µm
• Membrane filters remove
microbes >0.22 µm

Figure 7.4 37
Q&A

• In what situation is
filtration the only
practical way to
eliminate
undesirable
microbes?

38
Physical Methods of Microbial Control

• Low temperature has a bacteriostatic effect

• Refrigeration
• Deep-freezing
• Lyophilization (freeze drying)
• High pressure denatures proteins
• Desiccation: absence of water prevents metabolism
• Osmotic pressure uses salts and sugars to create
hypertonic environment; causes plasmolysis

39
Radiation

Figure 7.5 40
Radiation

• Ionizing radiation (X rays, gamma rays, electron

beams)
• Ionizes water to create reactive hydroxyl radicals (OH•)
• Damages DNA by causing lethal mutations
• Nonionizing radiation (UV, 260 nm)
• Damages DNA by creating thymine dimers
• Microwaves kill by heat; not especially antimicrobial

41
Check Your Understanding

• How is microbial growth in canned foods prevented? 7-

4
• Why would a can of pork take longer to sterilize at a
given temperature than a can of soup that also
contained pieces of pork? 7-5
• What is the connection between the killing effect of
radiation and hydroxyl radical forms of oxygen? 7-6

42
Chemical Methods of Microbial Control

Learning Objectives

• 7-7 List the factors related to effective disinfection.

• 7-8 Interpret the results of use-dilution tests and the
disk-diffusion method.

43
Principles of Effective Disinfection

• Concentration of disinfectant
• Organic matter
• pH
• Time

44
Use-Dilution Test

• Metal cylinders dipped in test bacteria are dried

• Dried cultures are placed in disinfectant for 10 min at
20°C
• Rings are transferred to culture media to determine
whether bacteria survived treatment

45
The Disk-Diffusion Method

• Evaluates efficacy of chemical agents

• Filter paper disks are soaked in a chemical and placed
on a culture
• Look for zone of inhibition around disks
Disk-Diffusion Method

Figure 7.6 47
Clinical Focus
• Which preparation is more effective?

Clinical Focus, p. 201 48

Check Your Understanding

• If you wanted to disinfect a surface contaminated by

vomit and a surface contaminated by a sneeze, why
would your choice of disinfectant make a difference? 7-
7
• Which is more likely to be used in a medical clinic
laboratory, a use-dilution test or a disk-diffusion test? 7-
8

49
Chemical Methods of Microbial Control

Learning Objectives
• 7-9 Identify the methods of action and preferred uses of
chemical disinfectants.
• 7-10 Differentiate halogens used as antiseptics from
halogens used as disinfectants.
• 7-11 Identify the appropriate uses for surface-active
agents.
• 7-12 List the advantages of glutaraldehyde over other
chemical disinfectants.
• 7-13 Identify chemical sterilizers.

50
Phenol & Phenolics

• Injure lipids of plasma membranes, causing leakage

Figure 7.7a, b
51
Bisphenols

• Contain two phenol groups connected by a bridge

• Hexachlorophene and triclosan
• Disrupt plasma membranes

Figure 7.7c, d 52
Triclosan

53
Biguanides

• Chlorhexidine
• Disrupt plasma membranes
• Used on skin, mucus membranes
• Surgical handscrubs

54
Chlorhexidine

55
Halogens

• Iodine
• Tinctures: In aqueous alcohol
• Iodophors: In organic molecules
• Alter protein synthesis and membranes
• Chlorine
• Oxidizing agents; shut down cellular enzyme systems
• Bleach: hypochlorous acid (HOCl)
• Chloramine: chlorine + ammonia

56
Halogens

57
Alcohols
• Denature proteins
and dissolves
lipids
• No effect on
endospores and
nonenveloped
viruses
• Ethanol and
isopropanol
• Require water

Table 7.6 58
59
Heavy Metals

• Ag, Hg, and Cu

• Silver nitrate may be used to prevent gonorrheal
ophthalmia neonatorum
• Silver sulfadiazine used as a topical cream on burns
• Copper sulfate is an algicide
• Oligodynamic action —very small amounts exert
antimicrobial activity
• Denature proteins

60
Oligodynamic action of heavy metals

61
Surface-Active Agents, or Surfactants

Soap Degerming; emulsification

Anions react with plasma

Acid-anionic detergents
membrane

Quarternary ammonium Bactericidal, denature

compounds proteins, disrupt plasma
(cationic detergents) membrane

62
Chemical Food Preservatives

• Sulfur dioxide prevents wine spoilage

• Organic acids
• Inhibit metabolism
• Sorbic acid, benzoic acid, and calcium propionate
• Control molds and bacteria in acidic foods and cosmetics
• Nitrites and nitrates prevents endospore germination

63
64
Antibiotics

• Bacteriocins—proteins produced by one bacterium that

inhibits another
• Nisin and natamycin prevent spoilage of cheese
Aldehydes

• Inactivate proteins by cross-linking with functional

groups (–NH2, –OH, –COOH, –SH)
• Used for preserving specimens and in medical
equipment
• Formaldehyde and ortho-phthalaldehyde
• Glutaraldehyde is one of the few liquid chemical sterilizing
agents

66
Gaseous Sterilants

• Used for preserving specimens and in medical

equipment
• Formaldehyde and ortho-phthalaldehyde
• Glutaraldehyde is one of the few liquid chemical sterilizing
agents

67
Plasma

• Fourth state of matter, consisting of electrically excited

gas
• Free radicals destroy microbes
• Used for tubular instruments

68
Supercritical Fluids

• CO2 with gaseous and liquid properties

• Use: Medical implants

69
Peroxygens

• Oxidizing agents
• Used for contaminated surfaces and food packaging
• O3, H2O2, peracetic acid

70
Check Your Understanding

• Why is alcohol effective against some viruses and not

others? 7-9
• Is Betadine an antiseptic or a disinfectant when it is
used on skin? 7-10
• What characteristics make surface-active agents
attractive to the dairy industry? 7-11

71
Microbial Characteristics and Microbial Control

Learning Objective

• 7-14 Explain how the type of microbe affects

the control of microbial growth.

72
Table 7.7 Effectiveness of Chemical Antimicrobials against Endospores and Mycobacteria
Microbial Characteristics

Figure 7.11 74
Histopathology in Figure 1 shows spongiform degeneration (indicated
by the blue pins) 75
Check Your Understanding

• The presence or absence of endospores has an

obvious effect on microbial control, but why are gram-
negative bacteria more resistant to chemical biocides
than gram-positive bacteria? 7-14

76
 Class discussion

Develop a list and group

the different microbial control
methods.

Answer should be based on the type of control measure,

group (physical or chemical) and if it kills, reduce numbers
or inhibits microbial growth.

78
 Case Study for discussion
As a standard procedure in a particular hospital, catheters are soaked in chlorine-based chemicals in between use for 30
mins. Despite this, it is reported this hospital had frequent cases of Urethral catheter-associated urinary tract infection
(CAUTI) after receiving treatment. To investigate the cause, a swab test on the catheters and patients’ bladder epithelial
cells, Uropathogenic Escherichia coli (UPEC) was detected.

Why didn’t the chemical disinfectant work to kill E. coli? Why is the organism present in all the samples tested?

Note that the catheters are also stored under anaerobic conditions to prevent oxygen supply needed for microbial growth.
Despite this, UPEC was still isolated.

What type of organism is UPEC based on its oxygen requirement? What solution would you suggest to adequately
sterilize the catheters?

Supporting Literature:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666759/pdf/ijms-18-02077.pdf

79
Case study for Discussion

Group 1 Why didn’t the chemical disinfectant work to kill E. coli?

Why is the organism present in all the samples tested?

What type of organism is UPEC based on its oxygen

Group 2 requirement? What solution would you suggest to
adequately sterilize the catheters?

State factors affecting ineffectiveness of control measures

Group 3
and suggest solutions to this.

List the mechanisms of microbial control, Pick the best

Group 4 solution to the problem experienced in this hospital from
this list and explain the mechanism of action of the
suggested control measures.
What Happens Next?
•In session 2 this week, we will look at Classification of
microorganisms and will discuss the Eukaryotic group

•This involves grouping of microbes based on their physiology and

structure, growth pattern and control. We will also look at the
different types of organisms classified as Eukaryotes.

•It is important that you engage with this content on myLMS,

BEFORE your next class.
 What’s next?
1. Review notes and readings for LO6.
2. Prepare readings for LO7.