A culture which contains a single species of microorganism

Process of increasing the population of microorganisms by providing their nutritional and physical requirements.

The extracellular substances which provide the cell with materials for building protoplasm and for energy generation.

Any nutrient material used for growth and cultivation of microorganisms in the laboratory.

Which of the following IS NOT a use of culture medium?

INDICATION OF POSITIVE AND NEGATIVE SEMI-SOLID SOLIDIFYING AGENT/AGAR

TYPES OF CULTURE MEDIA ACCORDING TO PHYSICAL STATE

Culture media used for isolating pure culture on a solid medium. It has no solidifying agent. 

TYPES OF CULTURE MEDIA ACCORDING TO PHYSICAL STATE

It contains 0.1 to 0.5% solidifying agent. This culture medium is used for motility testing.

TYPES OF CULTURE MEDIA ACCORDING TO PHYSICAL STATE

It contains 1.5 to 2.0% solidifying agent. This culture medium is used for growing large number of bacteria.

TYPES OF CULTURE MEDIA ACCORDING TO CHEMICAL COMPOSITION

All components are chemically defined.

TYPES OF CULTURE MEDIA ACCORDING TO PHYSICAL STATE

Not all components are chemically defined; no exact/definite amount of nitrogen mentioned.

TYPES OF CULTURE MEDIA ACCORDING TO PRINCIPAL FUNCTION, PURPOSE, AND APPLICATION

Can support most or almost all types of species

Nutrient Agar (NA) and Potato Dextrose Agar (PDA) are examples of what type of culture media?

TYPES OF CULTURE MEDIA ACCORDING TO PRINCIPAL FUNCTION, PURPOSE, AND APPLICATION

Distinguishes one type of bacteria from another. With special reagents like pH indicators or dyes. Differentiate one microorganism from another from the same family. 

Special indicators: Eosin Methylene Blue Agar (EMBA)

TYPES OF CULTURE MEDIA ACCORDING TO PRINCIPAL FUNCTION, PURPOSE, AND APPLICATION

Allows growth of a specific type of microorganism only. 

TYPES OF CULTURE MEDIA ACCORDING TO PRINCIPAL FUNCTION, PURPOSE, AND APPLICATION

Used to increase the number of microorganisms with unusual physiological characteristics. With special nutrients like blood. Example of this is blood agar.

TYPES OF CULTURE MEDIA ACCORDING TO PRINCIPAL FUNCTION, PURPOSE, AND APPLICATION

Of prescribed composition used for assay of vitamins, amino acids, and antibiotics. Assessment of biological activity of a substance. 

Used to determine the qualitative/quantitative production of such a compound by an organism.

Turbidity is measured:

Opaque if with growth vs Clear if free from growth

a macroscopically visible growth of cluster of microorganisms on a solid medium

ISOLATION TECHNIQUE

Types of Plating

Wire loop + pattern

The 1st and 3rd area must not overlap; a loop full of sample or 0.01 mL

The principle behind this is that as you go from one area to another, you clean the microorganisms. Hence STREAK

ISOLATION TECHNIQUES

Types of Plating

Surface only using l rod

ISOLATION TECHNIQUES

Type of Plating

Sample is pipetted first. No medium yet. 40 to 245 degrees Celsius heating. 

Molten agar to be mixed.

This results to surface and sub-surface colonies of microorganisms

PLATIING ISOLATION TECHNIQUE

ISOLATION TECHNIQUES

Isolation of specific types of microorganisms by a combination of nutrient and specific conditions

Used for unusual physiological types of microorganism which are present in small numbers and which grow slowly.

To grow to detectable level

ISOLATION TECHNIQUES

Used if the desired microorganism is present a higher level than any other microorganism

TO LESSEN

If too much sample is available, and you desire to go from higher to lower population.

ISOLATION TECHNIQUES

Uses a micropipette or a microscope to physically pick a single cell and transfer it on an agar medium./

Microelectrode to get single-cell.

ISOLATION TECHNIQUES

For samples with low population but liquid sample.

Uses a sterile membrane filter having a pore size that retains microorganisms

Membrane filter on a filter support . water sample is filtered through the membrane filter > membrane filter is then removed and placed in plate containing the appropriate medium > incubation for 24 hours > Typical Colonies

ISOLATION TECHNIQUES

Here, there is lower dilution in order to concentrate a sample.

STEPS IN PREPARING PURE CULTURES

Preserving Cultures aims to retain the viability and purity of the stock culture for a long period of time while maintaining its purity.

What are the 5 Methods of Culture Preservation?

In preservation of culture via periodic transfer to fresh media, we consider the following except:

If transfer is not quick and efficient, this might happen to culture.

CULTURE PRESERVATION METHODS 

It aims to limit the availability of oxygen in order to reduce the metabolic rate of microorganisms, thereby preserving them.

A disadvantage in overlaying cultures with mineral oil as a method of culture preservation is the fact that anaerobic microorganisms are immune to lower oxygen and not all microorganisms can have their metabolic rate reduced via lowering oxygen levels.

CULTURE PRESERVATION METHODS 

What is the alternative term for freeze-drying?

CULTURE PRESERVATION METHODS 

This method is advantageous for long-term survival and less opportunity for changes in the characteristics of culture. Also, storage containers used are small/compact. 

CULTURE PRESERVATION METHODS 

What is the temperature in freezing with liquid nitrogen?

CULTURE PRESERVATION METHODS 

In freezing with liquid nitrogen, what is added to protect the organism from extreme cold?

CULTURE PRESERVATION METHODS 

Samples are grown on sterile paper disk saturated with nutrients, then the disks are allowed to air dry and are stored aseptically (stored in a sterile environment). The temperature applied is 45 degrees Celsius.

Paper with agar: Sample is grown there

However, a limitation for this method is that it is only for spore- and cyst-formers

CULTURE PRESERVATION METHODS 

DRYING PROCESSES:

Drops of bacterial suspension in gelatin are placed on sterile plastic petri plates and dried off over __________ ________ under vacuum.

CULTURE PRESERVATION METHODS 

Drying Processes:

Bacteria in a small __________ are dried from liquid state using vacuum pump, desiccant, and water bath, 

CULTURE PRESERVATION METHODS 

Drying Processes:

Organisms are dried over _______ _______ in vacuum and stored in the ref.

Organizations which maintain authentic pure cultures of microorganisms. They provide ’type’ strains to microbiologists throughout the world.

National culture collection organization of the Philippines

CLICK PROCEED to proceed to LECTURE 4 Practice: CONTROL AND DESTRUCTION OF MICROORGANISMS

We control the growth of microorganisms for the following reasons:

1. Prevent spoilage of important commodities

2. Prevent infection

3. Prevent contamination of cultures, personnel, and environment

Kills bacteria

Inhibits bacterial reproduction and activity

Complete destruction of all microorganisms, including endospores. The environment is absolutely free from any microorganism.

Killing, inhibition, or removal of pathogenic microorganisms using disinfectants.

these are inanimate objects that harbor microorganisms

Examples of these are phones, pens, tables, and tablets; harboring microorganisms.

Reduction of microbial population to levels considered safe by public health standards.

To kill or inhibit microorganisms is the mode of action of sanitization

ANTISEPSIS VS ANTISEPTICS

Mechanical removal + use of mild chemicals on tissues or skin

PHYSICAL METHODS OF MICROBIAL CONTROL

This method and its mode of action is to denature proteins and other cellular components. Either moist or dry means.

MOA: Irreversible denaturation of enzymes and structural proteins. More effective in penetrating cells.

MOIST HEAT 

kills vegetative cells and eukaryotic spores only (some remain alive). Total sterility is NOT achieved.

MOIST HEAT

Uses autoclave or pressure cooker with 121 degrees Celsius or 15 PSI for 15 minutes. Water is subjected to this. 

What is being sterilized using steam under pressure?

For antibiotics, is steam under pressure used?

Indicator used to ensure sterility. Stripes show when proper sterilization is achieved.

Sterility indicator that is an alternative to the autoclave tape. It is an endospore former whose color changes if proper sterilization is observed; from yellow-orange to red-violet color.

Subject the material to 3 consecutive days to heat.

-Intermittent or fractional sterilization

-For materials destroyed beyond 100 degrees Celsius 

-Heating to 100 degrees Celsius for 15-30 minutes

-Done for 3 consecutive days

Tyndallization 3 Day Heating

Kill pathogens and reduce the number of spoilage in microorganisms. Developed by Louis Pasteur in the 1860s. It keeps intact the taste while microbes and pathogens are killed. If too much heat is applied, there will be denaturation and the food is no longer palatable. 

Pasteurization is based on this heat-resistant and fever-causing bacteria found in milk.

3 Modes of Pasteurization

Mode of Pasteurization

63 degrees Celsius for 30 minutes. 

If high heat = coagulation

Mode of Pasteurization

72 degrees Celsius for 15 minutes

Mode of Pasteurization

135 to 140 degrees Celsius for 1 to 2 secs

Used in green fresh milk or low fat milk

Sterilization vs Pasteurization

PHYSICAL METHODS OF MICROBIAL CONTROL

MOA: Incineration or burning to ashes

It uses alcohol lamps, Bunsen burner, Bacteri-cinerator

PHYSICAL METHODS OF MICROBIAL CONTROL

Used for cleaning pipettes

170 degrees Celsius for at least 2 hours

Mechanical convention oven

MOA: Oxidation of Molecular Components

Why is dry heating through hot air and direct flame longer and require higher temperature than moist heat?

Moist microbes have decreased metabolic activity at very ____ temperatures.

MOA: Decreased enzyme activity = Decreased metabolic activity under this temperature condition

MOA: Exclusion of microorganisms. 

-Common pore size is 0.2 um.

-For heat sensitive solutions

-What comes out is already sterile, while sterilized reservoir is left behind. Ex: antibiotics

-High efficiency particulate air (HEPA) filters 

1. Prevents Sample Contamination

2. Protects Personnel and Environment

-Filters air (membrane filter); uses laminar filter

MOA: Lowering of water activity (Aw) resulting to microbiostasis or microbiostatic. 

Removing moist

Water avaiable in a food sample that can be used by a microorganism

Modes of Desiccation

Increase salt/sugar to induce osmotic shock to microorganism. 

The increase in solutes such as salts and sugars induces osmotic shock, thereby lowered water activity via solutes

From 0.9 range of water percentage to 0.7 when microbes die due to osmotic shock

Shorter wavelength; strong penetrating power

MOA: Double Strand breaks in DNA; it can no longer replicate.

Gamma rays, X-rays, high-energy electron beam are used for materials that cannot be autoclaved. 

Ultraviolet Radiation with longer wavelength; lower energy

MOA: Formation of thymine dimers:

Proper bonding to bonding with the same strand (incorrect/improper bonding)

Thymine connects to thymine instead of adenine

With poor penetrating power - ”Surface Disinfection”

UV cannot penetrate paper or petri dish

For surface disinfection only

UV and visible light combination

MOA: Thymine dimers + Oxidation

Disadvantage: There is still air that can have microorganisms floating (suspended), but sunlight radiation kills microbes.

CHEMICAL METHODS OF MICROBIAL CONTROL

Natural or synthetic chemicals used to destroy or inhibit microorganisms

-cidal vs -static

PHENOLS AND PHENOLICS

CHEMICAL METHODS OF MICROBIAL CONTROL

MOA: Membrane (cell membrane) disruption and proteins denaturation. e.g. thymol, eucalyptol, cresols

An example of phenols and phenolics in which its mode of action is the inhibition of fatty acid-biosynthesis pathway. Banned by USFDA Sept 2016.

Present in hand soaps as anti-bacterial organic soaps

CHEMICAL METHODS OF MICROBIAL CONTROL

MOA: Protein denaturation. 

Can dissolve fats/lipids. 

Most effective form is at 70% concentration > just sufficient time to stay longer in hands and kill microorganisms

Alcohols are effective in enveloped viruses

CHEMICAL METHODS OF MICROBIAL CONTROL

Group VII A of the Periodic Table

CHEMICAL METHODS OF MICROBIAL CONTROL

HALOGENS

MOA: Oxidation of cellular components

Complexed to form iodophors which are for color.

Found in betadine

disinfectant used in iodine

CHEMICAL METHODS OF MICROBIAL CONTROL

HALOGENS

MOA: Oxidation of cellular components

Hypochlorous acid and sodium hypochlorite (bleach)

5.25% to 6.15% sodium hypochlorite

diluted to 1:100.

Recommended in disinfecting surfaces against the SARS-CoV-2 virus

CHEMICAL METHODS OF MICROBIAL CONTROL

HALOGENS

MOA: Interference of Metabolism

Active ingredient in toothpaste

CHEMICAL METHODS OF MICROBIAL CONTROL

MOA: Protein Denaturation.

Oligodynamic = even at low concentrations, it has the ability to kill microorganisms/bacterial population

It does not show selective toxicity

It indicates that even at low concentrations, this substance has the ability to kill bacterial population.

CHEMICAL METHODS OF MICROBIAL CONTROL

HEAVY METALS

Was used to treat syphilis

CHEMICAL METHODS OF MICROBIAL CONTROL

HEAVY METALS

Combined with antibiotics. Coating in medical supplies

CHEMICAL METHODS OF MICROBIAL CONTROL

HEAVY METALS

Used to control algal growth: with coatings (in socks to prevent smelling)

CHEMICAL METHODS OF MICROBIAL CONTROL

HEAVY METALS

Chloride, oxide, and pyrithione forms

CHEMICAL METHODS OF MICROBIAL CONTROL

HEAVY METALS: Zinc

Mouthwash

CHEMICAL METHODS OF MICROBIAL CONTROL

HEAVY METALS: Zinc

Antiseptic cream

CHEMICAL METHODS OF MICROBIAL CONTROL

HEAVY METALS: Zinc

Antidandruff shampoo and psoriasis and acne treatment

CHEMICAL METHODS OF MICROBIAL CONTROL

Lowers surface tension of liquids; physical removal of microorganisms.

Anionic vs Cationic

SURFACTANTS

CHEMICAL METHODS OF MICROBIAL CONTROL

In preservatives; flavorless, nontoxic, and readily metabolized

Applications: Food and Cosmetics

CHEMICAL METHODS OF MICROBIAL CONTROL

Organic Acids

Inhibition of cellular enzymes

CHEMICAL METHODS OF MICROBIAL CONTROL

Organic Acids

Oxidative phosphorylation and amino acid uptake interference

CHEMICAL METHODS OF MICROBIAL CONTROL

Organic Acids

Inhibition of metabolic enzymes

CHEMICAL METHODS OF MICROBIAL CONTROL

MOA: Nucleic acid and enzyme inactivation (attacks microbes and not hosts)

CHEMICAL METHODS OF MICROBIAL CONTROL

Alkylating Agents

Used in solution at a concentration of 37% (formalin). 

Storage of tissue specimens, embalming fluid, and in vaccine preparation.

CHEMICAL METHODS OF MICROBIAL CONTROL

Alkylating Agents

Two reactive aldehyde group

Found in surgical and medical equipment

CHEMICAL METHODS OF MICROBIAL CONTROL

Alkylating Agents

Sterilizing gas. Strong penetrating capability. Sterilization of items within plastics.

Used to sterilize syringes and catheters.

CHEMICAL METHODS OF MICROBIAL CONTROL

MOA: Oxidation of cellular components. Disinfectant/antiseptic

CHEMICAL METHODS OF MICROBIAL CONTROL

Peroxygens

Effective against bacteria, fungi, viruses, and endospores

Uses bubbling in which there is production of oxygen and water

CHEMICAL METHODS OF MICROBIAL CONTROL

Peroxygens

Acne medication solutions. Facial cleanser; cream

CHEMICAL METHODS OF MICROBIAL CONTROL

Peroxygens

Peroxygen in toothpaste

MEASURING MICROBIAL CONTROL

Effectiveness of Heat

Time needed to kill a given number of organisms at a specified temperature

The lower the ___________ _________, the more effective the mode.

MEASURING MICROBIAL CONTROL

Time required to destroy 90% of the organisms

^Temp = Shorter the value of this

MEASURING MICROBIAL CONTROL

Smallest amount of agent needed to inhibit the growth of a test organism.

At different concentration, take note which specific concentration inhibits microbe growth

In disc diffusion assay where antimicrobial compound is dispersed and the growth of microorganisms is inhibited, there are ______ __ ___________ measured.

FACTORS AFFECTING THE EFFECTIVENESS OF MICROBIAL GROWTH