Microbial Growth Chapter 4 And 5

Microbial Growth CHAPTER 4 and 5

Growth of Microbes • Increase in number of cells, not cell size • One cell becomes colony of millions of cells

Growth of Microbes • Control of growth is important for –infection control –growth of industrial and biotech organisms

Factors Regulating Growth • Nutrients • Environmental conditions: temperature, pH, osmotic pressure • Generation time

Chemical Requirements • #1 = water! • Elements – C (50% of cell’s dry weight) – Trace elements • Organic – Source of energy (glucose) – Vitamins (coenzymes) – Some amino acids, purines and pyrimidines

Nutritional Categories • Carbon sources – CO2 = – organic = • Energy sources – sunlight = – organic =

autotroph heterotroph phototroph chemotroph

A “Chemoheterotroph” would….. • Derive both carbon and energy from organic compounds

A “Chemoorganic autotroph would be…. Derives energy from organic compounds and carbon source from inorganic compounds

A related ancient group….. Lithoautotroph Neither sunlight nor organics used, rather it relies totally on inorganics

Nutritional Categories • Saprobe – lives on organic matter of dead organisms • Parasite – lives on organic matter of living host = pathogens

Environmental Factors Influencing Growth • • • • •

Temperature O2 pH Osmotic Pressure Others: radiation, atmospheric pressure

Temperature Optima • Psychrophiles: cold-loving • Mesophiles: moderate temperatureloving • Thermophiles: heat-loving • Each has a minimum, optimum, and maximum growth temperature

Fig. 7.8

Temperature Optima • Optimum growth temperature is usually near the top of the growth range • Death above the maximum temp. comes from enzyme inactivation • Mesophiles most common group of organisms • 40ºF (5°C) slows or stops growth of most microbes

Oxygen Requirements • Obligate aerobes – require O2 • Facultative anaerobes – can use O2 but also grow without it • Obligate anaerobes – die in the presence of O2

pH • Most bacteria grow between pH 6.5 and 7.5 • Acid (below pH 4) good preservative for pickles, sauerkraut, cheeses • Acidophiles can live at low pH

pH • Many bacteria and viruses survive low pH of stomach to infect intestines • Helicobacter pylori lives in stomach under mucus layer

Measuring Bacterial Growth

Bacterial Division • Bacteria divide by binary fission • Alternative means –Budding –Conidiospores (filamentous bacteria) –Fragmentation

Fig. 7.13

Generation Time • Time required for cell to divide/for population to double • Average for bacteria is 1-3 hours • E. coli generation time = 20 min –20 generations (7 hours), 1 cell becomes 1 million cells!

Fig. 7.14a

Plotting growth on graphs

Standard Growth Curve

Phases of Growth • Lag phase – making new enzymes in response to new medium • Log phase – exponential growth –Desired for production of products –Most sensitive to drugs and radiation during this period

Phases of Growth • Stationary phase – –nutrients becoming limiting or waste products becoming toxic – death rate = division rate • Death phase – death exceeds division

Measuring Growth • Direct methods – count individual cells • Indirect Methods – measure effects of bacterial growth

Fig. i7.6

Fig. 7.17

Turbidity

Metabolic Activity

Dry Weight

Metabolic Diversity in Bacteria *Bacteria can be placed into 2 groups based on how they obtain energy : 1. Heterotrophs – get energy by consuming organic molecules made by other organisms. *Most prokaryotes are heterotrophs. 2. Autotrophs – get energy by making their own food from inorganic molecules.

Types of Heterotrophs : A. Chemoheterotrophs – organisms that must take in organic molecules for both energy & carbon. B. Photoheterotrophs – organisms that are photosynthetic but need organic compounds as a carbon source.

Types of Autotrophs A. Photoautotrophs – organisms that use energy from sunlight to convert CO2 & water to carbon compounds. B. Chemoautotrophs – organisms that make organic molecules from CO2 using energy from chemical reactions.

Releasing Energy -Bacteria, like all organisms, release energy through cellular respiration, fermentation, or both. Cellular respiration – process that releases energy by breaking down glucose & other food molecules in the presence of oxygen (O2).

Fermentation – process by which cells release energy in the absence of O2.

Obligate aerobes vs. anaerobes -Organisms that require a constant supply of oxygen in order to live are called obligate aerobes, ex : Mycobacterium tuberculosis . -Organisms that must live in the absence of oxygen are called obligate anaerobes (oxygen would kill them), ex : Clostridium botulinum. -Organisms that can survive with or without oxygen are called facultative anaerobes, ex : E. coli. -Anaerobes perform fermentation to release energy & aerobes perform cellular respiration to release energy.

General Procedure of Bacteriological Diagnosis Specimens collection General rules The specimens should be representative of the infectious process; Sufficient material; Avoid the contamination of specimens; Be sent to the lab immediately in an appropriate method and examined ASAP. Be collected before using antimicrobial agents, e.g. antibiotics. 37

General Procedure of Bacteriological Diagnosis specimens Morphologic identification microscopy & staining

Isolation, identification Biochemical tests

Antigen detection EIA, ELISA, IF test, agglutination test

Serological diagnosis (Ab titer) convalescent phase / acute phase≥4

Molecular Biological Diagnosis(hybridization,

PCR, RT-PCR,etc)

38

• Anaerobic Medium a medium for the cultivation of certain anaerobes. The medium contains reducing agent, such as non-saturation fatty acid.

39

Based on the physical state • Liquid medium: – Without agar. – for the proliferation of bacteria.

• Solid medium: – 1.5-2.5% agar. – for the isolation and identification of bacteria – e.g., slant, Petri dishes/plates.

• Semisolid medium: – 0.3-0.5% agar. – for the observation of bacterial motility and preservation of bacteria.

40

Bacterial growth patterns • In liquid medium: Superficial growth; Turbidity/diffuse; Precipitate growing; (sediment)

 In solid medium: Confluent growth / Smear; Colony: a cluster of microorganisms growing on a solid medium. It is directly visible and arises from a single cell. 41

• General procedure of bacteriological Diagnosis • Culture medium

– concept – categories – bacterial growth patterns • Inoculation and transfer techniques

42

Inoculation and Transfer Techniques  Streak-plate technique  Slant inoculation  Liquid medium inoculation  Semisolid medium inoculation

43

Streak-plate technique four-area streak plate technique I

1/10

I

Rotate Flame plate loop 90 Rotate 90

II

1/5

Flame Rotate loop 90 III 1/4

IV 44

Slant inoculation

45

Liquid medium inoculation

46

Assignment #2 • Q1- Give account of dilution method for isolation of micro organism in pure culture