1. Inhibiting Microbial Growth in vitro
CLS 311: Basic Microbiology
Mrs. Amany Ahmed Niazy

2. Microbicidal or Microbistatic?
Microbicidal is the process or an agent that kills the microorganism.
The suffix -cidal or –cide means ''killing''.
Microbistatic
Microbistatic is the process or an agent that inhibits the growth and reproduction of the microorganism.
The suffix -static or –state means ''inhibiting or stopping''.

3. Cleansing
Cleansing  is the removal of organic material from instruments and equipment & may be done, clinically, in four steps:
Rinsing the object under cold water
Applying detergent and scrubbing object
Rinsing the object under warm water
Drying the object prior to sterilization or disinfection

4. Sterilization or Disinfection?
Sterilization is the complete destruction of all forms of microbial life including bacteria, viruses, fungi, parasites, and spores.
Disinfection
Disinfection is reducing or eliminating the number of pathogenic microorganisms to the point where they no longer cause disease. But this method does not affect spores.

5. Pasteurization and Sanitization
Pasteurization Is a disinfecting method used to eliminate pathogens from liquids e.g. milk, juice,.. Sanitization Is the use of chemical agents on food-handling equipment to meet public health standards and minimize chances of disease transmission e.g. use of hot water & soap in cleaning restaurants.

6. STERILIZATION AND DISINFECTION METHODS
Methods used to destroy or inhibit microorganisms are either physical or chemical, and sometimes both types are used.

7. Physical Methods

8. Physical Methods
They are commonly used in hospitals, clinics, and laboratories. Physical methods include:
Heat (Dray heat, Moist heat, Autoclave)
Cold.
Desiccation.
Radiation.
Ultrasonic waves.
Filtration.
Gaseous atmosphere.

9. Physical Method I- HEAT
Heat is considered the most common method for sterilization because it is practical, efficient, and inexpensive.
Heat kills microorganisms by denaturing their enzymes and other proteins.
There are three methods of sterilization or disinfection by heat:
dry heat
moist heat
autoclave.

10. Physical Method I- HEAT 1. Dry Heat
An effective way to sterilize metals, glassware, some powders, oils, and waxes.
Hot Air Oven:
It is done in a °C oven for 2hours or in °C oven for 1hour.
Burning (incineration):
Is used to destroy contaminated disposable materials.
Direct Flame:
Bunsen burner or electrical heating device is used to sterile wire loops and forceps used in the laboratory.

11. Dry heat Oven
Bunsen Burner
Electric Bunsen

12. Physical Method I- HEAT 2. Moist Heat
Is used to disinfect syringes, needles, and simple instruments by boiling (100°C) for 30 minutes.
Boiling is not always effective as
spores, like Mycobacteria, and
some viruses are not affected.

13. Physical Method I- HEAT 3. Autoclave
It is a large
metal chamber that uses
steam under pressure for
sterilization (like the
pressure cooker).
Autoclaving is done at 121.5°C and 15psi pressure for 20 minutes.
An autoclave tape or strip (commercially available) is used to ensure proper functioning.

15. Autoclave Tapes

16. Physical Method II- Cold
Freezing (below zero)
Freezing will greatly slow the metabolic activities of microorganisms leading to inhibition of their growth.
2. Refrigeration (4°C)
Refrigeration will slightly affect the metabolic activities of most microorganisms but it would not completely inhibit growth. It will slow down their growth.

17. Physical Method II- Cold
Slow Freezing is the most effective way as ice crystals that forms may rapture the cell membrane and cell wall of microbes.
CAUSION:
Thawing and refreezing of
food will allow the bacteria
and its spores to resume
growing.

18. Physical Method III- Desiccation (Drying)
Many microorganisms stay viable even after drying but they cannot reproduce i.e. desiccation will inhibit the growth of microorganisms.
When suitable moist and nutrient rich environment is available, the microorganism will grow rapidly.
It is often used for preservation of food, antisera, antitoxin, antibiotics, and pure culture of microorganisms.

19. Keep in mind that dried viable pathogens may be found in blood, pus, fecal material and dust that are found on floors , in bedding, on clothing. Thus wet mopping of floors, damp dusting of furniture rolling bed linens an towels carefully, is very important to avoid transmission of these microbes through air.

20. Physical Method IV- Radiation
There are three different types of radiation that can be used to control microorganisms:
Ionizing radiation.
Ultraviolet radiation.
Microwave radiation.

21. Electromagnetic spectrum

23. Physical Method IV- Radiation A. Ionizing Radiation (Sterilize)
Gamma rays, X-rays, and Beta rays from radioactive materials have short wavelengths (less than 1 nanometer)
Cause death or mutations in microorganisms as they damage the DNA and proteins.
Used to sterilize heat-sensitive materials including medical equipment, disposable surgical supplies and drugs. Radiation can be carried out after packaging.
Irradiation has been used for many years to control microorganism on herbs, vegetables, meat.
Is it safe to humans working, and consuming these products??!

26. Physical Method IV- Radiation B
Physical Method IV- Radiation B. Ultraviolet light (Non-ionizing Radiation) (disinfect)
Wavelength is longer than 1 nanometer  low penetration.
Damages DNA which cause mutations or death.
Most effective on actively multiplying organisms.
Endospores are the most UV-resistant.
Most commonly used as UV-lamps that disinfect to destroy microbes in the air or drinking water and to disinfect surfaces.
Disadvantages: Damages skin, eyes and does not penetrate paper, glass, and cloth.

27. UV-Lamps

28. Physical Method IV- Radiation C. Microwave Radiation
Do not affect microorganisms directly, but kill microbes by the heat they generate in an item.
Organisms often survive microwave cooking, however, because the food heats unevenly.
May kill microbial cells in moist foods but not spores.

29. Physical Method V- Ultrasonic Waves
Used for cleaning delicate equipments.
Ultrasonic cleaners consist of water tanks, were short sound waves pass
through, where it mechanically
remove organic debris.
Eequipment must then be washed
and sterilized before use.

30. Physical Method VI- Filtration
Filtration is the removal of microbes by passage of a liquid or gas through a screen like material with small pores.
Filters of various pore sizes can be used.
Used to sterilize heat sensitive materials like vaccines, enzymes, antibiotics, and some culture media.
Air Filtration:
High Efficiency Particulate Air Filters (HEPA):
Used in operating rooms and burn units to remove bacteria from air.
Used in biological safety cabinets (laminar flow hoods)

32. Physical Method VI- Filtration HEPA

33. Physical Method VII- Gaseous Atmosphere
In rare cases, changing the atmosphere can be used as a way to inhibit the growth of microorganisms.
Aerobes and microaerophiles can be killed by placing them in oxygen-free atmosphere. Whereas, anaerobes can be killed by placing them in oxygen atmosphere.

34. Chemical Methods

35. skin and mucous membranes. objects, surfaces, and floors.
Chemical Methods
Antiseptics
An antiseptic is a chemical
agent that is used to inhibit
microbial growth on human
skin and mucous membranes.
Disinfectants
A disinfectant is a chemical
agent that is used to inhibit
microbial growth on inanimate
objects, surfaces, and floors.

36. How to Select an Appropriate Chemical???
Toxicity  what are the risk to humans or environment.
Activity in the presence of organic matter  some chemicals are inactivated by organic matter.
Compatibility with the material being treated  some corrosive like hypochlorite can damage some metals and rubber.
Cost and availability.
Storage and stability  some have limited shelf life.
Concentration of disinfectant lose effectiveness when diluted more than the manufacturer recommendation.
Number and type of microorganism to be killed (or inhibited)

37. Groups of Chemicals Used as Disinfectant
Example
Uses
Phenol & Phenol Derivatives
Lysol
Widely used to disinfect inanimate objects.
Alcohols
Ethanol or Isopropanol
Antiseptics for skin
Disinfectant for treating instruments.
Halogens
Chlorine & Iodine
Povidone Iodine are used as antiseptics.
Solutions of chlorine are widely used to disinfect surfaces, and water.
Aldehydes
Formaldehyde and Gluteraldehyde
Formalin to preserve biological specimens.
To preserve viruses in vaccines.
Peroxygens
Hydrogen peroxide
Personal care products, like lotions, deodorant soaps.

39. Resistance to Killing
Gram-negative bacteria (with their outer membrane) are generally more resistant than gram-positive bacteria to disinfectants and antiseptics
Stationary-phase (I.e., non-growing) bacteria generally are more resistant than log-phase (I.e., growing) bacteria
Mycobacteria, endospores, and protozoan cysts and oocysts are very resistant to disinfectants and antiseptics
Nonenveloped viruses are generally more resistant than enveloped viruses to disinfectants and antiseptics
Organic matter (such as vomit and feces) frequently affects the actions of chemical control agent
Disinfectant activity is inhibited by cold temperatures
Longer application times are preferable to shorter

42. Those humans are starting to annoy me