Syllabus Outline till Christmas

Week Date /Time Event Reading

9 26/9 Lecture 1 Unit Introduction/History of Chapter 1
Microbiology
10 3/10 Lecture 2 Microscopes and Microbial Cell Chapter 1 and 2
Structure
11 10/10 Lecture 3 Cell structure and function Chapter 2

All dates 12 14/10 Lecture 4 Microbial nutrition Chapter 3 (15

and times further reading)
12 17/10 (9-10, 11-12, Laboratory introduction and training Online Lab
are 2-3,) Manual
13 24/10 (9-11,12-2, 3- Laboratory 1 Lab Manual
subject to 5,)
25/10 Chapter 5
13 Lecture 5 Microbial Growth and Control
change-
15 7/11 (9-11, 12-2, 3-5,) Laboratory 2 Lab Manual
please
check your 16 14/11 (9-11,12-2, 3-
5,)
Laboratory 3 Lab Manual

portal! 18 28/11 (9-11,

3-5)
12-2, LAB EXAM Lab Manual

20 9/12 Lecture 6 Viruses Chapter 8

Formative MCQ assessment online

Emailing me about missing a lab
Yes -please do- use your University
Account
There are no make up sessions unless you
have an ECF approved.
ECF process- go to your moodle site
 Control of
Microbial Growth
Control of microbial growth Bacteriocidal Total cell count

Log cell number

Degrees of Inhibition
Viable
cell count

Bacteriocidal Time
Bacteriolytic
Bacteriolytic
Fungicidal

Log cell number

All result in death of the organism Total cell count

Bacteriostatic Viable
cell count
Fungistatic Time
Prevent or inhibit growth
Bacteriostatic

Log cell number

Total cell count

Viable cell
count

Time
Terminology
• Aseptic surgery techniques
 Sterilization: Removal of all microbial life
 Commercial sterilization: C. botulinum endospores
 Disinfection: Removal of pathogens
 Antisepsis: Removal of pathogens from living tissue
 Sanitization: Lower microbial counts on eating utensils
 Biocide/Germicide: Kills microbes
 Bacteriostasis: Inhibiting, not killing, microbes
• Bacterial populations die at a constant logarithmic rate.

Figure 7.1a
 Effectiveness of Antimicrobial Treatment
• Depends on:
– Number of microbes
– Environment (organic
matter, temperature,
biofilms)
– Time of exposure
– Microbial
characteristics

Figure 7.1b
 Actions of Microbial Control Agents
• Change membrane permeability
• Damage to proteins
• Damage to nucleic acids
Physical Methods of Microbial Control
1. Dry Heat

2. Moist heat

3. Radiation

4. Filtration
Dry Heat
• Dry heat sterilization kills by oxidation
– Flaming
– Incineration
– Hot-air sterilization
– Less effective than moist heat

Hot-air Autoclave
Equivalent treatments 170˚C, 2 hr 121˚C, 15 min
 Moist Heat
• Moist heat coagulation of proteins
• Autoclave: Steam under pressure

https://www.youtube.com/watch?v=uDgY64bux2k
Fig. 27-3
Chamber
pressure
gauge
Steam exhaust
Steam
exhaust
valve

Door Jacket chamber

Thermometer
and valve
Air exits through vent

Steam supply
valve
Steam enters here

Autoclave time
130
Stop
steam
Temperature (°C)

120

Begin Sterilization time

110 pressure

Flowing Temperature Temperature

steam of object being of autoclave
100 sterilized

0 10 20 30 40 50 60
Total cycle time (min)
Steam Sterilization
• Steam must contact
surface.
• Expulsion of all air
• Usually 15-20mins

Figure 7.3
 Heat
• Heat
– Thermal death point (TDP): Lowest temperature at
which all cells in a culture are killed in 10 min.
– Thermal death time (TDT): Time to kill all cells in a
culture at specific ºC
Decimal
reduction

Survival fraction (log scale)

100
time (D)
50°C
Decimal Reduction Time 10
(DRT) -Minutes –kill 90% 70°C

at a given temperature 1
60°C

0.1
10 20 30 40 50
Time (min)
 Pasteurization
• Pasteurization reduces spoilage organisms and
pathogens
• Equivalent treatments
– 63°C for 30 min
– HTST: 72°C for 15 sec
– UHT: 140°C for <1 sec
– Thermoduric organisms survive- why isn’t this a
problem?
– Tyndallisation
 Radiation
• Radiation damages DNA
– Ionizing radiation (X rays, gamma rays, electron beams)
– Nonionizing radiation (UV)
– Microwaves kill by heat
Table 27-1
Filtration
Filtration physically removes microbes
a) Glass fibre, b) membrane, c) nuclepore
Nucleopore=different size pores for specific exclusion
Fig. 27-8
 Chemical Methods of Microbial Control
• Joseph Lister- phenol- 1867
• Principles of effective disinfection
– Concentration of disinfectant
– Organic matter
– pH
– Time
Ignaz Semmelweis 1818-1865

Hungarian obstetrician
Puerperal fever
30% death rate
Autopsy room
Chlorinated lime
Death rate – 1%
 Types of Disinfectants
• Phenol
• Phenolics: Lysol
• Bisphenols:
Hexacholorphene,
Triclosan
– Disrupt plasma
membranes
– Stable and good with
organics
Types of Disinfectants
• Biguanides: Chlorhexidine
– Disrupt plasma membranes

 Halogens: Iodine, chlorine

 Oxidizing agents
 Bleach is hypochlorous acid (HOCl)

http://www.periodictable.com/Elements/019/index.html
Types of Disinfectants
• Alcohols: Ethanol,
isopropanol
– Denature proteins,
dissolve lipids

Tortora Table 7.6

Types of Disinfectants
• Heavy metals: Ag, Hg, and Cu
– Oligodynamic action
– Denature proteins
Types of Disinfectants
• Chemical food preservatives
– Organic acids
• Inhibit metabolism
• Sorbic acid, benzoic acid, and calcium propionate
• Control molds and bacteria in foods and cosmetics

– Nitrite prevents endospore germination- nitrosamines

– Antibiotics. Nisin and natamycin -cheese
Types of Disinfectants
• Gaseous sterilants
– Denature proteins
– Ethylene oxide

 Peroxygens
 Oxidizing agents
 O3, H2O2, peracetic acid
 Chemical Methods of Microbial Control
• Evaluating a disinfectant
– Disk-diffusion method

Tortora Figure 7.6

Microbial Characteristics and Microbial Control

Figure 7.11 Tortora

 Microbiological
Laboratory Safety

http://www.hse.gov.uk/biosafety/biologagents.pdf
 Biohazard
• An agent of biological origin that can
cause disease in humans
– Microorganism
– Toxin
– Allergen
 Agents and Risks
• The “agent” is the what creates risk

• Risks to the worker or environment are often

unknown

• Determining “acceptable risk”?

Guidelines for Microorganism Use
•Laws and regulations
•HSE -Health and Safety Executive
•CDC -Biosafety in Microbiological and Biomedical
Laboratories (BMBL)
•WHO -(World Health Organization) Biosafety Manual
Biosafety
The combined use of
 laboratory practices,
 laboratory facilities and
 safety equipment to work with
potentially infectious m/organisms.

To protect:
 Workers / Students
 Products / Experimental results
 Environment / Laboratory classroom
Microorganism Categories
Microorganisms can be categorized in a number of
ways.
i) Morphology
ii) Genetics
iii) By tissues that they infect – Enterobacteriaceae

Or

iv) Pathogenicity and communicability- BioSafety Level

(BSL 1-4)
Containment (CL) and Biosafety Levels
(BSL) – USA and European Directive
• Different than the Risk Groups!!
– Risk groups used in risk assessment
– CL/BSL are used in risk management
• CL/BSL are ways to control the agent
– facilities, safety equipment, practices, PPE, etc.
• Once risk is assessed then the appropriate CL/BSL is
determined
 Containment or Biosafety Levels
• CL-1/BL-1: agents are not known to cause disease

• CL2/BL-2: agents are associated with human disease

• CL3/BL-3: agents are associated with human disease and

are potentially transmitted as aerosols

• CL4/BL-4: agents of life threatening nature

Containment /Biosafety Level 1 (BL-1)
Standard Work Practices
• Use mechanical pipetting devices
• Wash hands frequently
• Minimize splashes and aerosols
• Decontaminate work surfaces daily
• Handle wastes properly

Personal Protective Equipment (PPE)

 Lab coat or apron- Gloves as needed
http://www.cafepress.co.uk

 Safety glasses or goggles

Containment CL-2 / Biosafety Level 2 (BL-2)
Use CL2/ BL-2 practices when working with:
• Agents of moderate potential hazard to personnel and the
environment
• Basic lab, but restricted access, containment during certain
processes (i.e. aerosols, large volumes, etc.)
• Autoclave and Biological Safety Cabinet desired
• Use good laboratory practices, waste disposal, and aseptic
techniques

• Example: most non-respiratory, non lethal, agents

• Human blood or body fluids
• E. coli 0157:H7
• Clostridium botulinum
• Retroviral vectors
• Human cells in cell culture
 Containment / Biosafety Level 2 (BL-2)
Standard Work Practices PPE
• Use mechanical pipetting
devices  Lab coat or apron
• Wash hands frequently  Safety glasses or goggles
• Minimize splashes and  Gloves
aerosols
 Biosafety cabinet
• Decontaminate work
surfaces daily  Aerosols or splashes
• Handle wastes properly  Large volumes
 High concentrations
Bioaerosols
 Containment / Biosafety Level 2 (BL-2)

• Adequate illumination
• Eyewash facility
• Negative air pressure
• Autoclave available
• Biological safety cabinet
• Lab must be separated from public areas
Containment /Biosafety Level 3
• Agents of high hazard to personnel or environment
• Respiratory exotic or indigenous agents which are
easily transmissible causing severe to fatal disease
in humans, but, for which treatments exist
• All work is contained, engineering controls and
controlled environments.
Example: Mycobacterium tuberculosis, Salmonella
typhi, Bacillus anthracis, SARS, etc.
 Containment/ Biosafety Level 4
• Too scary for me !!!
• Hemorrhagic fever, Marburg, Ebola, deadly agents
with no cure.
• Total containment, airtight labs, “submarine” doors,
air pumps, water treatment, HEPA filtration, etc.
• Positive pressure “moonsuits”
Seams, joints, and doors are sealed to make the building airtight.
Air does not flow in or out under the doors.
Air is pumped through HEPA filtration system
All air ducts are welded stainless steel and tested to be airtight.
Inside many BSL-4 facilities -buffer corridors that help protect the
laboratories in the event of a bombing attack from outside.
Airlocks, fumigation chambers, disinfectant "dunk tanks," and
waste water treatment systems ensure that absolutely everything
is decontaminated.
http://www.niaid.nih.gov
Aug 2019
• Airtight, pressurized suits have dedicated life-support systems
that include redundant breathing air compressors, alarms and
emergency backup air tanks, and a HEPA air filtration systems

• Biosafety cabinets serve as additional barriers protecting the

worker and the environment.
• Researchers in biocontainment suits must shower with strong
disinfectant before leaving the lab and removing the suit.
http://www.niaid.nih.gov
http://www.youtube.com/watch?v=4sYSyuuLk5g
Laboratory Acquired Infections (LAI)
Bacterial:
• 76% from clinical labs
• 8% from research labs
Viral:
• 16% from clinical labs
• 70% from research labs
• 32% from animal related activities
Exposure:
60% acquired from inhalation

Other exposures include: digestion, sharps, splashes,

direct and indirect contact