Medical Microbiology Microbial Metabolism Dr. Oruba lec. 4

Microbial Metabolism

                 Growth of Bacteria How do bacteria grow?

Goals What are the stages of cell growth? What is happening in cells during cell growth? What are the effects of environmental conditions on cell growth?

Binary Fission

Bacterial Doubling Time Escherichia coli 20 minutes Mycobacterium tuberculosis 18 hours Mycobacterium leprae 14 days

Growth :is the increase in all components of bacteria (cell wall ,cell membrane …. etc). The signs that indicate the bacterial growth are:- Turbidity :which can be detected by naked eyes or spectrophotometry. Color changes :some microorganisms are capable of producing pigments . Gas production :some bacteria produce gasses that can be collected in inverted tubes or it can push the bacterial growth outside. Change in pH :due to acid production by bacteria .  

Terms related to growth :-   Terms related to growth :- Viable cell count (cell concentration) :the number of bacteria that are able to grow ,this is determined by serial dilution method ,measured by (count ⁄ ml) Cell mass :it is the dry weight of bacteria which obtained after centrifuge. Total cell count :the same of viable cell count but it contains dead microorganism. Generation time :time required for development of two dells from single one by binary fission (1→2→4→8→…..etc) ,which is divided into three types : Fast growing :generation time (G.T.) is few hours . Slow growing :G.T. is 12-24 hours . Medium growing .

Growth and Growth curve: Generation time; is the time required for a single living cell to divide into two cells Doubling time ; is the time required for the whole cells in the culture to double its number. Growth rate ; it is the number of generations per unit of time, which is equal to; Growth rate = O.693 / Generation time  

What is happening at each stage? 1- Adaptation (lag) phase; it is the time required for cell to adapt to the new conditions and that by synthesizing all the necessary compounds, enzymes and pathways, which become able to utilize the new components of the new medium that the cells have been transferred to.  2- Phase of positive acceleration; it is the phase at which the bacteria have completed all its requirements and become ready for growth and multiplication. 3- Exponential (logarithmic, log) phase; it is the phase at which the bacteria grow and multiply at its maximum, under the experimental conditions. During this phase also, the storage granules increase, and more than one nucleoid may be seen. 4- Phase of negative acceleration; due to the fast growth of the bacteria in the log phase, several factors will be accumulated that affect the growth, such as:

*decrease in Oxygen concentration * decrease or exhaustion of one or more of the medium components * increase in CO2 concentration * increase in concentration of end products 5- Stationary phase; it is the phase during which, the number of cells dies is almost equal to the number of cells formed by division per unit of time. 6- Decline or Death phase; due to continued bad conditions that facing the bacteria, the number of cells dies per unit of time is much higher than the cells formed by division until no living cell will remain in the culture.  

What kinds of questions can we ask using growth experiments? 1-What is the effect of temperature on growth? 2-What is the effect of pH on growth? 3-What is the effect of Oxygen on growth? 4 -What is the effect of Enzymes on growth?

Effect of temperature on growth Figure: 06-16 Caption: Effect of temperature on growth rate and the molecular consequences for the cell. The three cardinal temperatures vary by organism.

Temperature Figure 6.1

Classification of organisms according to their need of Temperature: 1- Psychrophiles: are those organisms that live in temperatures with an average between 5 ـــــ -20oC 2- Mesophiles: are those organisms that live in temperatures with an average between 15 ـ 45 oC 3- Thermophiles: are those organisms that live in temperatures with an average between 40 ـــ 85 oC Optimum temperature; it is the temperature at which we obtain maximum growth rate, at the experiment conditions.

Maximum growth temperature: it is the highest temperature at which cells can grow, higher than this temperature cells will not grow or die. Minimum growth temperature; it is the lowest temperature at which cells can grow below this temperature cells will not grow, or die.

3- H-ion Concentration pH range, optimum pH Majority of pathogenic bacteria grow best at neutral or slightly alkaline pH (7.2 – 7.6) . Lactobacilli require acidic pH Vibrio cholerae require alkaline pH

The Effect of Oxygen (O2) on Growth Table 6.1

*Formation of toxic materials such as H2O2, OH-, O-2…….   Classification of organisms according to their need of Oxygen: Obligate aerobes: are those organisms that live only in the presence of oxygen with its atmospheric concentration. Obligate anaerobes: are those organisms that live only in the complete absence of oxygen, its presence will lead to death of these organisms. Facultative Anaerobes; are those organisms that live in the presence or absence of oxygen, but the presence oxygen will inhibit the anaerobic system Microaerophiles; are those organisms that live aerobically, but under low concentration of oxygen. Aerotolerants; are those group of microorganisms that normally live and grow in the absence of oxygen, but O2 presence will lead to stop its growth without killing it.

What are the effects of Oxygen on growth? Many forms of life require oxygen for _________________. aerobic respiration Some forms of oxygen can actually be toxic! Metabolism, UV light, chemical reactions can create toxic forms of oxygen Aerobes have enzymes to detoxify toxic forms of oxygen.

Catalase Activity Negative Positive Catalase activity is easily detected by dropping hydrogen peroxide onto a bacterial colony and observing the appearance, or lack thereof, of effervescence as a result of oxygen gas generation. We, ourselves, have often detected catalase activity in our own cells when we clean wounds using hydrogen peroxide.

Peroxidase Also catalyzes the breakdown of hydrogen peroxide Differs from catalase - does not produce oxygen H2O2 + 2 H+ 2 H2O ex. Streptococcus Some organisms lack catalase but neutralize hydrogen peroxide using a different enzyme, peroxidase. In this case, peroxide is combined with two protons to form two molecules of water but without the concomitant production of oxygen gas. One example of an organism that uses a special peroxidase is Streptococcus which grows well in oxygen yet lacks catalase.

Anaerobic Culture Methods Reducing media Contain chemicals (thioglycollate or oxyrase) that combine O2 Heated to drive off O2

Anaerobic Culture Methods Anaerobic jar Figure 6.5

Transport of nutrients: Uptake of nutrients… Two factors should be known for a particular cell’s need for a certain chemical compound. ability of that compound to pass through cytoplasmic membrane ability of the cell to use that compound as a source of energy or building block…  

Factors affecting Transport; Temperature, pH, substrate concentration, presence or absence of analogues, toxins, other obstacles (that react with cell membrane proteins and lipids), also heavy metals…. etc.. These factors affect all types of cells whether they are unicellular organisms or tissue cells. *there are three types of transport of nutrients known in living organisms:   Passive (simple) diffusion; - the driving force is the concentration gradient on both sides of the cellular membrane…. transport is from high concentration to low concentration, and stops when the concentration becomes equal on both sides of the membrane… this process is used for transport of water , dissolved O2, CO2, and also lipid soluble materials such as ethanol and glycerol…

2- Facilitated diffusion: it is common in eukaryotic cells, and present only in few prokaryotic cells… substrates are transported by the help of a special carrier proteins or permeases.. mechanism is unknown, but a conformational change occur in the carrier protein… substrate –carrier complex is moving from high concentration to low concentration, and stops when the concentration of the substrate becomes equal on both sides of the membrane.. 3- Active Transport: There are certain general characteristics of the active transport:

a complex is formed "carrier-substrate complex " on the external surface of the membrane…. Requires an excess of energy…. For increasing or decreasing the affinity of substrate to protein carrier…these proteins (or enzymes) are synthesized only in the presence of the substrate….sometimes, certain prokaryotic cells posses the complete set for the metabolism of the substrate inside the cell, but cannot metabolize it from the environment, these are called "cryptic mutants"…. Transport occurs against concentration gradient, i.e. from low concentration to high concentration… No modification occurs in substrates during the transport process… Posses a high degree of specificity, mainly for sugars, but may share the same carrier in case of certain amino acids..

References: 1- Jawetz, Melnick, & Adelberg’s.( 2013). Medical Microbiology (Twenty-Sixth Edition). 2- Kenneth Todar. (2008).Todar’s Online Textbook of Bacteriology ,University of Wisconsin.

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