Increase in number of cells, not cell size Populations Colonies Microbial Growth Increase in number of cells, not cell size Populations Colonies

Culture Media Culture medium: Nutrients prepared for growth of microorganisms 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 Slants: large surface area for growth Deeps: show oxygen requirements Plates: short term use

Types of Media Chemically defined media: exact chemical composition is known (autotrophic bacteria) Complex media: chemical composition may vary and includes nutrients including extracts from yeast, meat or plants (heterotrophic bacteria)

Selective and Differential Media Selective Media: suppress growth of unwanted bacteria and encourage growth of desired bacteria. Differential Media: distinguish colonies of desired organism from others growing on the same plate

Macconkey Agar- Selects for Gram negative Differentiates between bacteria that ferment lactose Mannitol Salt Agar- Selects for Gram Positive Differentiates between bacteria that ferment mannitol (sugar alcohol) turn mannitol to acid causes color change

(selects for gram-) (lactose fermentation)

Nutrient Agar Commonly used for routine cultivation of bacteria

Biosafety Levels 1: No special precautions 2: Lab coat, gloves, eye protection 3: Biosafety cabinets to prevent airborne transmission 4: Sealed, negative pressure Exhaust air is filtered twice

Biosafety Level 4 (BSL-4) Laboratory

Live Cell Cultures Viruses are too small to view without special equipment that is very expensive In the lab, we innoculate live cell cultures with the viral specimen We examine the culture tubes for patterns of cell destruction by the virus (cytopathic effect or CPE)

Selective Media By inhibiting unwanted organisms with salts, dyes, or other chemicals, selective media allow growth of only the desired microbes.

Differential Media Make it easy to distinguish colonies of different microbes. pH is always Monitored to Ensure optimal Growth conditions (most grow best At pH 7)

Enrichment Culture Encourages growth of desired microbe Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria Inoculate phenol-containing culture medium with the soil, and incubate Transfer 1 ml to another flask of the phenol medium, and incubate Only phenol-metabolizing bacteria will be growing

Obtaining Pure Cultures A pure culture contains only one species or strain A colony is a population of cells arising from a single cell or spore or from a group of attached cells A colony is often called a colony-forming unit (CFU) The streak plate method is used to isolate pure cultures

The Streak Plate Method Each streak dilutes the amount of bacteria until individual colonies can be seen

Preserving Bacterial Cultures Deep-freezing: –50° to –95°C Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum

Direct Methods of Measuring Cell Growth A standard plate count Filtration Most probable number (MPN) method Direct microscopic count

Serial Dilutions In order to more accurately count the colonies on the plates, the original sample is often diluted to reduce the total number of bacteria into a more “countable” range If the original 1 ml of sample has 10,000 bacteria, we would dilute it until it was around 100 bacteria that is a more countable number on a plate That plate # would be multiplied by the dilution factor to obtain the total original count of bacteria

Serial Dilutions The 1:100,000 dilution was plated, incubated, & then counted to obtain 18 bacteria What is the original # of bacteria? 1,800,000/ml

Plate Counts # of viable microbes and assumes that each bacterium grows into a single colony

Plate Counts After incubation, count colonies on plates that have 25–250 colonies (CFUs)

Counting Bacteria by Membrane Filtration bacteria are retained on the surface of a membrane filter and then transferred to a culture medium to grow and subsequently be counted.

Most Probable Number Multiple tube MPN test Count positive tubes used for microbes that will grow in a liquid medium it is a statistical estimation.

Most Probable Number Compare with a statistical table.

Direct Microscopic Count The microbes in a measured volume of a bacterial suspension are counted with the use of a specially designed slide.

Direct Microscopic Count

Estimating Bacterial Numbers by Indirect Methods Spectrophotometer Measuring the metabolic activity Measuring dry weight

Turbidity Spectrophotometer is used to determine turbidity by measuring the amount of light that passes through a suspension of cells.

Turbidity

Metabolic activity estimating bacterial numbers is measuring the metabolic activity of the population for example, acid production or oxygen consumption

Measuring Dry Weight For filamentous organisms such as fungi, measuring dry weight is a convenient method of growth measurement.