Unit III

Dr. Pabba Shivakrishna

Nutritional requirements and types of media and growth

conditions…..

Nutritional Requirements of Cells

• Every organism must find in its environment all of the substances required for energy generation and cellular biosynthesis.

• The chemicals and elements of this environment that are utilized for bacterial growth are referred to as nutrients or nutritional requirements.

• Many bacteria can be grown the laboratory in culture media which are designed to provide all the essential nutrients in solution for bacterial growth.

The Major Elements

• At an elementary level, the nutritional requirements of a bacterium such as E. coliare revealed by the cell's elemental composition, which consists of C, H, O, N, S. P, K, Mg, Fe, Ca, Mn, and traces of Zn, Co, Cu, and Mo. These elements are found in the form of water, inorganic ions, small molecules, and macromolecules which serve either a structural or functional role in the cells.

The Requirements for Growth

PHYSICAL REQUIREMENTS

– Temperature– pH– Oxygen– Hydrostatic Pressure– Osmotic pressure

CHEMICAL REQUIREMENTS (NUTRITIONAL FACTORS)

– Carbon– Nitrogen, sulfur, and

phosphorous– Trace elements– Oxygen– Organic growth

factor

1) pH

• Optimum pH: the pH at which the microorganism grows best (e.g. pH 7)

• Most bacteria grow between pH 6.5 and 7.5• Molds and yeasts grow between pH 5 and 6• According to their tolerance for acidity/alkalinity,

bacteria are classified as:Acidophiles (acid-loving): grow best at pH 0.1-5.4Neutrophiles: grow best at pH 5.4 to 8.0Alkaliphiles (base-loving): grow best at pH 7.0-11.5

Physical Factors Required for Bacterial Growth

2) Temperature• According to their growth temperature range, bacteria can be

classified as:Psychrophiles : grow best at 15-20oCPsychrotrophs: grow between 0°C and 20–30°CMesophiles : grow best at 25-40oCThermophiles : grow best at 50-60oC

Typical Growth Rates and Temperature– Minimum growth temperature: lowest temp which species can

grow – Optimum growth temperature: temp at which the species grow

best– Maximum growth temperature: highest temp at which grow is

possible

Food Preservation Temperatures

3) Oxygen• Aerobes: require oxygen to grow• Obligate aerobes: must have free oxygen for aerobic respiration (e.g.

Pseudomonas)• Anaerobes: do not require oxygen to grow• Obligate anaerobes: killed by free oxygen (e.g. Bacteroides)• Microaerophiles: grow best in presence of small amount of free oxygen• Capnophiles: carbon-dioxide loving organisms that thrive under

conditions of low oxygen• Facultative anaerobes: carry on aerobic metabolism when oxygen is

present, but shift to anaerobic metabolism when oxygen is absent• Aerotolerant anaerobes: can survive in the presence of oxygen but do

not use it in their metabolism

• Obligate: organism must have specified environmental condition• Facultative: organism is able to adjust to and tolerate environmental

condition, but can also live in other conditions

Patterns of Oxygen Use

5) Osmotic Pressure

• Environments that contain dissolved substances exert osmotic pressure, and pressure can exceed that exerted by dissolved substances in cells

• Hyperosmotic environments: cells lose water and undergo plasmolysis (shrinking of cell)

• Hypoosmotic environment: cells gain water and swell and burst

Plasmolysis

Halophiles• Salt-loving organisms which require moderate to large

quantities of salt (sodium chloride)

• Membrane transport systems actively transport sodium ions out of cells and concentrate potassium ions inside

• Why do halophiles require sodium?1) Cells need sodium to maintain a high intracellular

potassium concentration for enzymatic function2) Cells need sodium to maintain the integrity of their

cell walls

Responses to Salt

The Great Salt Lake in Utah

Chemical Requirement: Nutritional Factors

1. Carbon sources2. Nitrogen sources3. Sulfur and phosphorus4. Trace elements (e.g. copper, iron, zinc, and

cobalt)5. Vitamins (e.g. folic acid, vitamin B-12,

vitamin K)

Chemical Requirements

• Carbon– Structural organic molecules, energy source– Chemoheterotrophs use organic carbon sources– Autotrophs use CO2

Chemical Requirements• Nitrogen

– In amino acids and proteins– Most bacteria decompose proteins– Some bacteria use NH4+ or NO3–

– A few bacteria use N2 in nitrogen fixation

Chemical Requirements

• Sulfur– In amino acids, thiamine, and biotin– Most bacteria decompose proteins– Some bacteria use SO4

2– or H2S

• Phosphorus – In DNA, RNA, ATP, and membranes– PO4

3– is a source of phosphorus

Chemical Requirements

• Trace elements– Inorganic elements (mineral) required in small

amounts– Usually as enzyme cofactors– Ex: iron, molybdenum, zinc

• Buffer– To neutralize acids and maintain proper pH– Peptones and amino acids or phosphate salts may

act as buffers

Organic Growth Factors

• Organic compounds obtained directly from the environment

• Ex: Vitamins, amino acids, purines, and pyrimidines

Preparation of Culture Media

• Culture medium: Nutrients prepared for microbial growth

• Sterile: No living microbes• Inoculum: Introduction of microbes into

medium• Culture: Microbes growing in/on culture

medium

Agar

• Complex polysaccharide • Used as solidifying agent for culture media in

Petri plates, slants, and deeps• Generally not metabolized by microbes• Liquefies at 100°C• Solidifies at ~40°C

CULTURE MEDIA USED IN MICROBIOLOGY

Definition, purpose/importance

History of culture media

Classification of culture media

Growth pattern of bacteria

Microbiological culture Method of cultivating microbial

organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions.

Louis Pasteur used simple broths made up of urine or meat extracts. Robert Koch realized the importance of solid media and used potato pieces to grow bacteria. It was on the suggestion of Fannie Eilshemius, wife of Walther Hesse (who was an assistant to Robert Koch) that agar was used to solidify culture media.

History of culture medias

Before the use of agar, attempts were made to use gelatin as solidifying agent. Gelatin had some inherent problems…. It existed as liquid at normal incubating temperatures (35-37oC) Digested by certain bacteria

Continued….

Agar Used for preparing solid medium Obtained from seaweeds. No nutritive value Not affected by the growth of the bacteria. Melts at 98oC & sets at 42oC 2% agar is employed in solid medium

During typical bacteria growth (growth cycle) bacteria cell divide by binary fission and their mass and number increase in an exponential manners. Bacterial growth in culture can be separated into at least four distinct phases.

Bacterial Growth Curve

Bacterial Growth Curve

The Lag Phase• Organisms do not increase significantly in number

• They are metabolically active

• Grow in size, synthesize enzymes, and incorporate molecules from medium

• Produce large quantities of energy in the form of ATP

The Log Phase

• Organisms have adapted to a growth medium

• Growth occurs at an exponential (log) rate

• The organisms divide at their most rapid rate

• a regular, genetically determined interval (generation time)

Synchronous growth: A hypothetical situation in which the number of cells in a culture would increase in a stair-step pattern, dividing together at the same rate

Nonsynchronous growth: A natural situation in which an actual culture has cell dividing at one rate and other cells dividing at a slightly slower rate

1) Cell division decreases to a point that new cells are produced at same rate as old cell die. 2) The number of live cells stays constant.

Decline (Death) Phase1) Condition in the medium become less and less

supportive of cell division2) Cell lose their ability to divide and thus die3) Number of live cells decreases at a logarithmic

rate

Stationary Phase

Measuring Microbial Growth

Direct Methods

• Plate counts• Filtration• MPN• Direct microscopic

count

Indirect Methods

• Turbidity• Metabolic activity• Dry weight

Types of culture media

I. Based on their consistency a) solid mediumb) liquid mediumc) semi solid medium

II. Based on the constituents/ ingredientsa) simple mediumb) complex mediumc) synthetic or defined mediumd) Special media

Special media Enriched media Enrichment media Selective media Indicator media Differential media Transport media

III.Based on Oxygen requirement- Aerobic media- Anaerobic media

Solid media – contains 2% agar Colony morphology, pigmentation, hemolysis can be

appreciated. Eg: Nutrient agar, Blood agar

Liquid media – no agar. For inoculum preparation, Blood culture, for the isolation

of pathogens from a mixture. Eg: Nutrient broth

Semi solid medium – 0.5% agar. Eg: SIM

Simple media / basal media - Eg: NB, NA- NB consists of peptone, yeast extract, NaCl, - NB + 2% agar = Nutrient agar

Complex media Media other than basal media. They have added ingredients. Provide special nutrients

Synthetic or defined media Media prepared from pure chemical substances

and its exact composition is known Eg: peptone water – 1% peptone + 0.5% NaCl in

water

Enriched media

Substances like blood, serum, egg are added to the basal medium.

Used to grow bacteria that are exacting in their nutritional needs.

Eg: Blood agar, Chocolate agar

Blood agar Chocolate agar

Enrichment media Liquid media used to isolate

pathogens from a mixed culture. Media is incorporated with

inhibitory substances to suppress the unwanted organism.

Eg: Selenite F Broth – for the isolation of

Salmonella, Shigella Alkaline Peptone Water – for Vibrio

cholerae

Selective media The inhibitory substance is added to a solid

media.Eg: Mac Conkey’s medium for gram negative

bacteria TCBS – for V.cholerae LJ medium – M.tuberculosis Wilson and Blair medium – S.typhi Potassium tellurite medium – Diphtheria bacilli

TCBSMac Conkey’s medium

Potassium Tellurite media LJ media

Indicator media These media contain an indicator which

changes its colour when a bacterium grows in them.

Eg: Blood agar Mac Conkey’s medium Christensen’s urease medium

Urease medium

Differential media A media which has substances incorporated in

it enabling it to distinguish between bacteria. Eg: Mac Conkey’s medium Distinguish between lactose fermenters & non

lactose fermenters.

Lactose fermenters – Pink colonies Non lactose fermenters – colourless colonies

Transport media Media used for transporting the

samples. Delicate organisms may not

survive the time taken for transporting the specimen without a transport media.

Eg: Stuart’s medium – non nutrient soft

agar gel containing a reducing agent Buffered glycerol saline – enteric

bacilli

Anaerobic media These media are used to grow anaerobic organisms. Eg: Robertson’s cooked meat medium, Thioglycolate

medium.

Growth in Continuous Culture

• A “continuous culture” is an open system in which fresh media is continuously added to the culture at a constant rate, and old broth is removed at the same rate.

• This method is accomplished in a device called a chemostat.

• Typically, the concentration of cells will reach an equilibrium level that remains constant as long as the nutrient feed is maintained.

Basic Chemostat System

Our Chemostat System

Serial dilution Method of Bacterial Enumeration

Many studies require the quantitative determination of bacterial populations. The two most widely used methods for determining bacterial numbers are:I. The standard plate count method.II. Spectrophotometer (turbid metric) analysis.

The standard plate count method is an indirect measurement of cell density ( live bacteria).

The spectrophotometer analysis is based on turbidity and indirectly measures all bacteria (cell biomass), dead and alive.

Introduction

The Plate Count (Viable Count)

However, if the sample is serially diluted and then plated out on an agar surface in such a manner that single isolated bacteria form visible isolated colonies, the number of colonies can be used as a measure of the number of viable (living) cells in that known dilution.

The number of bacteria in a given sample is usually too great to be counted directly.

Keep in mind that if the organism normally forms multiple cell arrangements, such as chains, the colony-forming unit may consist of a chain of bacteria rather than a single bacterium.

In addition, some of the bacteria may be clumped together. Therefore, when doing the plate count technique, we generally say we are determining the number of Colony-Forming Units (CFUs) in that known dilution.

By extrapolation, this number can in turn be used to calculate the number of CFUs in the original sample.

bacterial counts by these methods are usually expressed as colony forming units per milliliter (CFU/mL).

Normally, the bacterial sample is diluted by factors of 10 and plated on agar.

After incubation, the number of colonies on a dilution plate showing between 30 and 300 colonies is determined.

A plate having 30-300 colonies is chosen because this range is considered statistically significant.

If there are less than 30 colonies on the plate, small errors in dilution technique or the presence of a few contaminants will have a drastic effect on the final count. (too few to count (TFTC). Likewise, if there are more than 300 colonies on the plate, there will be poor isolation and colonies will have grown together. (too numerous to count (TNTC).

Procedure

1) Using sterile technique, transfer 1 mL sample to the first dilution blank. Mix the bottle by inverting it 20 times. Label the bottle "10-1."

2)Using a fresh pipette, transfer 1 mL from the first blank to the second blank. Mix as before. Label the second bottle "10-2." 3) Using a fresh pipette, transfer 1 mL from the first blank to the second blank. Mix as before. Label the second bottle "10-2“.

4) Using a fresh pipette, transfer 1 mL from the first blank to the third blank. Mix as before. Label the second bottle "10-3“.

5) Using a fresh pipette, transfer 1 mL from the first blank to the forth blank. Mix as before. Label the second bottle "10-4“.

6) Using a fresh pipette, transfer 1 mL from the first blank to the second blank. Mix as before. Label the fifth bottle "10-5“.

7) Label the Petri dishes: 10-2, 10-3, 10-4, 10-5, and 10-6, respectively.

8) transfer liquid from the dilution blanks to the Petri dishes. Use a separate pipette for each blank, not for each plate (i.e. if more than one plate uses liquid from a single blank, a single pipette may be used for that blank).

9) One at a time, add a tube of molten nutrient agar to each Petri dish. After adding the agar, gently swirl the dishes in pattern for 30 seconds to mix the bacteria with the agar.

10)After the agar has thoroughly solidified, incubate the plates at 37°C for 24 to 48 hours.

11)Count the number of colonies on a plate that has between 30 and 200 colonies. Any plate which has more than 200 colonies is designated as "too numerous to count" (TNTC). Plates with fewer than 30 colonies do not have enough individuals to be statistically acceptable.

Colonies Forming Units {CFU}

Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor multiplied by the amount of specimen added to agar plate.

To compute the number of CFU/mL, use the formula: c = concentration, CFU/mL n = number of colonies d = dilution blank factor s = volume transferred to plate.

CFU Calculation Example

You count 46 colonies on your plate You put 1 ml of bacterial culture into 99 ml of

saline and plated 0.1 ml Dilution 1/100CFU= 46

1/100 * 0.1 = 46 * 100 * 10 =46000 CFU/ml