CONTROL OF

MICROBIAL
GROWTH
FARHANAH AHMAD SHUHAIMI
CENTRE FOR DIETETICS STUDIES
FACULTY OF HEALTH SCIENCES
UNIVERSITI TEKNOLOGI MARA
TERMS decontamination
process
• Sterilization vs. Disinfection
– Sterilization
• destroying all forms of life
• heat, ethylene oxide gas, hydrogen peroxide gas, plasma, ozone, and radiation
• Steam Sterilization (Autoclaving)
– Autoclaving (saturated steam under pressure of approximately 15 psi to achieve a
chamber temperature of at least 121oC for a prescribed time) rapidly achieves
destruction of microorganisms, decontaminates infectious waste and sterilizes
laboratory glassware, media, and reagents.
– Disinfection
• Destroying pathogens or unwanted organisms
• Disinfection reduces the level of microbial contamination.
• Chemical disinfection does not kill spores, unlike chemical sterilisation.
• Some common laboratory disinfectants include freshly prepared 10% bleach and 70%
ethanol.
– Chemical Germicide Activity Level:
• High Level Disinfection
• Intermediate Level Disinfection
• Low Level Disinfection
AUTOCLAVE
• Disinfection
1. High Level Disinfection
– Comprises high concentrations of chemical germicides (ex: concentrated sodium hypochlorite).
alive and replicate

– Kills vegetative microorganisms and inactivates viruses

– Does not kill high numbers of bacterial spores
– High level disinfectants are typically used for short time periods (10-30 min) for disinfection purposes, but
may achieve sterilization if left in contact with the surface for long time periods (6-10 hours).
– Not for use on environmental surfaces like floors or lab benches
• treatment of medical devices and dental instruments

2. Intermediate Level Disinfection

– Kills vegetative microorganisms, including Mycobacterium tuberculosis, all fungi, and inactivates most
viruses
– May be used for housekeeping and disinfection of laboratory benches
3. Low Level Disinfection
– Kills most vegetative bacteria, some fungi, and inactivates some viruses.
– Does not kill M. tuberculosis
Tb

– Also known as “hospital disinfectants” or “sanitizers”

TERMS
• Disinfectant vs. Antiseptic
not for human
– Disinfectant being
only for surfaces

• antimicrobial agent used on inanimate objects

– Antiseptic
lower concentration

minimal toxicity
• antimicrobial agent used on living tissue
BACTERICIDAL VS. BACTERIOSTATIC

• Bactericidal - kills bacteria

• Bacteristatic - inhibits bacterial growth
• Fungicidal -kill
keep in
stationary growth

• Fungistatic inhibit
growth

• Algacidal
• Algastatic
 FACTORS THAT EFFECT
ANTIMICROBIAL ACTIVITY
danger : 4 60 °

1. Temp zone

2. Time
3. Concentration of Antimicrobial agent
some microbe survive

4. Type of Microbe
can

in absence of O2

5. Activity of Microbe
6. Presence of organic matter
TARGETS OF ANTIMICROBIAL
AGENTS
1. Cell membrane
2. Enzymes & Proteins
3. DNA & RNA
METHODS TO CONTROL
MICROBIAL GROWTH
temp
pH

1. Physical osmotic pressure

2. Chemical water
PHYSICAL METHODS USED TO CONTROL MICROBIAL
GROWTH

• 1. Heat
– Moist Heat
• Boiling Water
• Steam Heat (Autoclave)
– Dry Heat
• Direct Flaming
• Incineration
• Hot Air Sterilization (Oven)
• 2. Filtration very fine
filter

• 3. Radiation
– Ionizing Radiation
– Non-Ionizing Radiation
• 4. Pasteurization (Heat)
 PHYSICAL METHODS OF MICROBIAL CONTROL
• 1. Heat
– works by denaturing enzymes and proteins
– A. Thermal Death Point (TDP)
• lowest temperature at which all microorganism in a liquid culture are killed in 10 minutes

– B. Thermal Death Time (TDT)

• minimum length of time in which all microorganisms in a liquid culture are killed at a given
temperature
MOIST HEAT

• 1. Boiling Water
– kills vegetative bacterial cells, Fungi and many viruses
– not effective for endospores and some viruses
– Hepatitis (20 min)
• Moist heat at 98°C for 1 minute will partially inactivate HBV.
• Incubation at 60°C for 10 hours (pasteurisation) will also inactivate HVB
– Some spores may survive boiling water for up to 20 hrs
MOIST HEAT

• 2. Autoclave (Steam under pressure)

– preferred method of sterilization
– Water boils at 100 oC
– Increasing the pressure raises the Temp.
– 15 psi/ 1 atm ------> 121 oC
– 121 oC for 15-20 min.
DRY HEAT

• 1. Direct Flaming
– Inoculating Loop and Needle 100% effective
• 2. Incineration
– disposable wastes (paper cups, bags,
dressings)
• 3. Hot Air Sterilization
– Oven ( 170 0C for 2 hours)
– used on substances that would be damaged
by moist heat sterilization
• dressings or powders
FILTRATION
 Removes microorganisms from solutions that might be damaged by heat
 Example – membranes of cellulose acetate, polycarbonate and variety of plastic
materials.
◦ culture media
◦ enzymes
◦ vaccines
◦ antibiotics
RADIATION

• Ionizing Radiation
– gamma rays & x-rays
• penetrates most substances

• Used on substances that could be damaged by heat

– plastic petri dishes
– plastic syringes
– surgical gloves
RADIATION

• 2. Non-Ionizing Radiation
– UV Light
• does not penetrate plastic, glass or proteinaceous matter
• Used to reduce microbial populations
– hospital rooms
– nurseries
– operating rooms
 PASTEURIZATION
• Disinfection - not sterilization (disinfectant only removes unwanted organisms)
• Pasteurization involves heating liquids at high temperatures for short amounts of time.
• Pasteurization kills harmful microbes in milk without affecting the taste or nutritional value
• Kills harmful bacteria that can lead to diseases like listeriosis, typhoid fever, tuberculosis,
diphtheria and brucellosis
• Mycobacterium tuberculosis
– 63 0C for 30 minutes
– 72 0C for 15 seconds
– Thermodurics
• able to survive high temperature
CHEMICAL ANTIMICROBIALS IN FOOD
- SYNTHETIC
• Organic acid – acetic, benzoic, Lactic, Propionic, sorbic.
• Nitrites – usually in meat.
• Parabenzoic acid – beers, noncarbonated soft drinks, fruit-based beverages
• Sodium Chloride (salt)
• Phosphate
• Sulfites – fruits, fruits juices, wines, sausages, meats.
NATURALLY OCCURING
ANTIMICROBIAL
 INTRODUCTION
• Antimicrobial are sometimes called preservatives.
• Major antimicrobial target is spoilage microorganism and foodborne pathogen.
• Spoilage – causes discoloration, produce slime in food (can warn people not to
bubble
eat)
• Pathogens can grow without obvious signs of spoilage.
• The cost of natural antimicrobial is low compared to chemical preservatives.
1. LYSOZYMES
• Produced in eggs, milk and eggs. form
powder

• Commercial lysozyme is made from dried egg white.

• Lysozymes in hen eggs has 129 amino acids.
• Stable heat (100 0c) and at low pH (<5.3).
• Inactivate at low temperatures and when pH is increased.
• The enzyme breaks the β – 1,4 glycosidic bonds between N-
acetylmuramic acid and N-acetylglucosamine of peptidoglycan in
the bacteria.
• Most active against Gram positive bacteria.
LYSOZYMES
• Inhibits:
• C. botulinum, C.thermosaccharolyticum, C.tyrobutyricum, B.
Stearothermophilus, B. cereus and Listeria monocytogenes.
• Lysozyme is the main antimicrobial in egg albumen.
• Less effective in Gram –ve. – can be increased by
chelators (eg EDTA). EDTA enhance lysozyme activity
by helping it penetrates to the peptidoglycan.
 LYSOZYMES – USES IN FOOD
natural preservative

• To prevent gas formation in cheese.

• Preserve seafood, vegetables, pasta and salads.
2. LACTOFERRIN AND OTHER IRON-
BINDING PROTEINS
• Milk and eggs contain antimicrobial iron-binding proteins.
• Lactoferrin – main in milk
– The antimicrobial activity of LF is mostly due to two mechanisms.
1. The first is iron sequestration in sites of infection, which deprives the
microorganism of this nutrient, thus creating a bacteriostatic effect.
2. The other mechanism is the direct interaction of the LF molecule with the
infectious agent. The positive amino acids in LF can interact with anionic
molecules on some bacterial, viral, fungal and parasite surfaces, causing cell lysis.
• Microbes with low iron requirement s are not inhibited by lactoferrin. (Iron
stimulates growth in many genera, including Clostridium, Escherichia, Listeria,
Pseudomonas, Salmonella, Staphylococcus,Vibrio and Yersinia).
3. AVIDIN
• Egg albumen protein.
• Makes up 0.05% of the total eggs albumen protein.
• Stable to heat and a wide pH range.
• Binds biotin (vitamin B7) at a ratio of four biotin molecules per avidin
molecules. Biotin activates enzymes in some metabolic cycles.
• Avidin inhibits bacteria and yeast that require biotin for growth.
• Avidin also binds transport protein in the E-coli outer membrane – inhibiting
bacteria by interfering with trasnport system.
4. SPICES AND THEIR ESSENTIAL
OILS
• Ancient Egyptians used spices for food preservation – cinnamon, oregano,
rosemary etc.
• Have active chemical components and required concentration, the organism
are sensitive to them. (NOT all spices have antimicrobial activity).
• Most phenolic compound may interfere the membrane function.
SPICES AND THEIR ESSENTIAL OILS
Spice Antimicrobial compound Concentration Target activity

Cloves Eugenol B. subtilitis, E.coli, Salmonella,

L.monocytogenes, S.aures.
Cinnamon Cinnamon aldehyde
10-20% volatile oil
Oregano Calvacrol MIC 0.02-0.05%; E-coli, Salmonella, L.
MBC 0.03-0.1% monocytogenes, S.aureus,
Thyme Thymol B.cereus, some yeast and
molds.

Rosmary Terpenes, borneol, piene, MIC 0.3%; Gram positive bact – B.

camphor, thujone MBC 0.5% cereus, S.aureus, L.
monocytogenes
Vanillin 4-hydroxy-3methoxy- 500-2000µg/ml Molds and non lactic gram
benzaldehyde +ve bact.

MIC - minimum inhibitory conc.

MBC – minimun bactericidal conc.
5. ONIONS AND GARLIC
• Best characterized plant antimicrobial
• Inhibits B. subtilis, Serratia marcescens, Mycobacterium spp., B.cereus, C.
botulinum type A, E-coli, Lactobacillus plantarum, Leuconostoc mesenteroids,
Salmonella spp., Shigella spp. And S. aureus.
• Fungi – Aspergillus flavus, Aspergillus parasiticus, candida albicans, and
Cryptococcus penicilium, Rhodotorula, Saccharomyces,Torulopsis and
Trishosporon.
• Garlic – allicin, inhibits sulfhydryl containing enzymes in bacteria.
• Onions – protocatehuic acid and catechol
-
6. ISOTHIOCYANATES
• Potent antimicrobial.
• Made from glucosinolates in plant cells from the cruciferae,
or mustard family (cabbage, mustard, kale, turnips,
cauliflower, brocoli etc).
• Fungi, yeast and bacteria are inhibited by 0.016 – 0.062
µg/ml in vapor phase or 10 to 600 µg/ml in liquid.
• Isothiocyanates inhibits cells by reacting with disulfide
bonds or in activating sulfhydryl enzymes.
7.PHENOLIC COMPOUND

• Have an aromatic ring with one or more hydroxyl groups.

• Classified as simple phenols, phenolic acids, hydroxycinnamic acids and
flavonoid.
• Simple phenolic compound include monophenols, diphenols
(hydroquinone), triphenols (gallic acid).
• Only simple phenols used as preservatives in wood smoke.
• Smoking meats, cheeses, fish and poultry imparts both falvors and
preservative effect.
• Several commercial smoke preparation at 0.25 and 0.5% reduce
L.monocytogenes.
THAN K YOU