The document discusses principles of bacterial growth through binary fission and how bacteria grow in nature through biofilms. It also covers microbial growth in the laboratory through obtaining pure cultures and following growth curves, and how environmental factors like temperature, oxygen, pH and water affect microbial growth.
The document discusses principles of bacterial growth through binary fission and how bacteria grow in nature through biofilms. It also covers microbial growth in the laboratory through obtaining pure cultures and following growth curves, and how environmental factors like temperature, oxygen, pH and water affect microbial growth.
The document discusses principles of bacterial growth through binary fission and how bacteria grow in nature through biofilms. It also covers microbial growth in the laboratory through obtaining pure cultures and following growth curves, and how environmental factors like temperature, oxygen, pH and water affect microbial growth.
The document discusses principles of bacterial growth through binary fission and how bacteria grow in nature through biofilms. It also covers microbial growth in the laboratory through obtaining pure cultures and following growth curves, and how environmental factors like temperature, oxygen, pH and water affect microbial growth.
AND IMMUNOLOGY OPT 416 HS246 Dr MAIMUNAH MUSTAKIM 2
INTRODUCTION TO MICROBIOLOGY 3
LEARNING OBJECTIVES AT THE END OF THE LESSON, STUDENTS ARE ABLE TO EXPLAIN OR IDENTIFY: I. Principles of microbial growth by binary fission. II. Bacterial growth in nature - biofilms. III. Role of Biofilms in human health. IV. Microbial Growth in Laboratory – pure colonies, aseptic technique, growth curve. V. Microbial Growth factors: temperature, oxygen, pH, water. VI. Detection of Microbial Growth. VII. Bacterial Pathogenicity and Virulence 5
Principles of Bacterial Growth
• Prokaryotic cells divide by binary fission • One cell divides into two • Two into four, six, eight and so forth. 7
• Prokaryotic cells divide by binary fission
• One cell divides into two • Two into four etc. • Cell growth is exponential • pattern of balanced growth where all the cells are dividing regularly by binary fission. • Exponential growth has important health consequences • Generation time • Time it takes for population to double • a.k.a. doubling time • Varies among species Principles of Bacterial Growth • Growth can be calculated • Nt = N0 x 2n • (Nt ) number of cells in population • (N0 ) original number of cells in the population • (n) number of divisions • Example • N0 = 10 cells in original population • n = 12 • 4 hours assuming 20 minute generation time • Nt = 10 x 212 • Nt = 10 x 4,096 • Nt = 40,960 Binary Fission 10
Bacterial Growth in Nature
• Conditions in nature have profound effect on microbial growth • Changes in the environment are detected by cells. • Synthesize compounds useful for growth • Cells produce multicellular associations to increase survivability • Example • Biofilms - any group of microorganisms in which cells stick to each other and often also Biofilm layer to a surface • Slime layers - extracellular material that surrounds bacteria cells Bacterial Growth in Nature • Biofilm • Formation begins when planktonic bacteria attach to surfaces • Other bacteria attach and grow on initial layer • Has characteristic architecture (structure) • Contains open channels for movement of nutrients and waste • Cells within biofilms can cause disease • Treatment becomes difficult – the structure resists immune response and antimicrobials • Bioremediation (the use of microorganisms (bacteria, fungi) to break down contaminants is beneficial use of biofilm 13
LIFE CYCLE OF BIOFILMS
1 2 3 4 5
Developmental stages in biofilm formation.
1. One or more planktonic bacterial species adhere to a biotic/abiotic surface. 2. Attached bacteria grow as a multicellular community by the formation of slime layers. 3. Microcolonies formed in which they multiply and mature. 4. This microbial infrastructure results in the development of a mature biofilm. 5. Biofilms serve as bacterial reservoirs that are transmitted back to the environment through biofilm dispersal and then colonize new surfaces. MAIMUNAH MUSTAKIM SEPT 2011 14 15
Role of Biofilm in Humans Health
16 DEVELOPMENT OF BIOFILM IN MEDICAL DEVICES AND HUMAN TISSUE 17 18 19
Biofilms on Medical Devices 20
Biofilms in Human Infections Biofilm formation in Catheter
https://youtu.be/97vX_jDhSqM
Clinical Microbiology and Infection 2015 21S1-S25DOI: (10.1016/j.cmi.2014.10.024)
22
Ventilator associated pneumonia (VAP)
causes prolonged intubation and a prolonged stay in ICU with the associated costs. It is also a serious cause of mortality in compromised patients.
A combination of oral care treatments
(brushing and suctioning) in a proper oral care protocol means care providers can efficiently prevent oropharyngeal bacterial biofilm build- up. This in turn leads to a reduction of VAP and aspiration pneumonias and greatly reduces associated treatment costs 23
https://youtu.be/aj2w0wequyQ
https://www.intersurgical.com/info/ oralcare Bacterial Growth in Nature • Interactions of mixed microbial communities • Prokaryotes live in mixed communities • Many interactions are cooperative • Waste of one organism nutrient for another • Some cells compete for nutrient • Synthesize toxic substance to inhibit growth of competitors 25
Microbial Growth in Laboratory
Obtaining Pure Culture • Pure culture defined as population of cells derived from single cell • All cells genetically identical • Cells grown in pure culture to study the activities of specific species • Pure culture obtained using special techniques • Aseptic technique • Minimizes potential contamination
• Cells grown on culture media
• Can be broth (liquid) or solid form Obtaining Pure Culture • Culture media can be liquid or solid • Liquid is broth media • Used for growing large numbers of bacteria • Solid media is broth media with addition of agar • Agar marine algae extract • Liquefies at temperatures • Bacteria grow in colonies on above 95°C solid media surface • Solidifies at 45°C • All cells in colony descend • Remains solid at room from single cell temperature and body • Approximately 1 million cells temperature produce 1 visible colony Obtaining Pure Culture • Streak-plate method • Simplest and most commonly used in bacterial isolation • Object is to reduce number of cells being spread • Solid surface dilution • Each successive spread decreases number of cells per streak 29 Culture Techniques Aseptic Technique •Physical means of preventing contamination • Protective clothing • Safe use of equipment • Sterilize loops • Avoid creation of aerosols • Class II safety cabinets • HEPA filter Aseptic Technique
•Inoculation of media • Agar plate • Agar slant • Indicator/selective medium Culture Techniques • Observing culture after 24 hours • Colony characteristics • Presence (or absence) of hemolysis Bacterial Growth in Laboratory Conditions
• Cells in laboratory grown in closed or batch
system • No new input of nutrient and no release of waste • Population of cells increase in predictable fashion • Follows a pattern called growth curve Bacterial Growth in Laboratory Conditions
• The Growth Curve
• Characterized by five distinct stages • Lag stage • Exponential or log stage • Stationary stage • Death stage • Phase of prolonged decline Bacterial Growth in Laboratory Conditions • Lag phase • Number of cells does not increase • Cells prepare for growth • “Tooling up”
• Log phase • Period of exponential growth • Doubling of population with each generation • Produce primary metabolites • Compounds required for growth • Cells enter late log phase • Synthesize secondary metabolites • Used to enhance survival • Antibiotics Bacterial Growth in Laboratory Conditions
• Stationary phase • Overall population remains relatively stable • Cells exhausted nutrients • Cell growth = cell death • Dying cell supply metabolites for replicating cells
• Death phase • Total number of viable cells decreases • Decrease at constant rate • Death is exponential • Much slower rate than growth Bacterial Growth in Laboratory Conditions
• Phase of prolonged decline
• Once nearly 99% of all cells dead, remaining cells enter prolonged decline • Marked by very gradual decrease in viable population • Phase may last months or years • Most fit cells survive • Each new cell more fit than previous Bacterial Growth in Laboratory Conditions
• Colony growth on solid medium
• In colony, cells eventually compete for resources • Cells grow exponentially and eventually compete for nutrients • Position within colony determines resource availability • Cells on edge of colony have little competition and significant oxygen stores • Cells in the middle of colony have high cell density • Leads to increased competition and decreased availability of oxygen Bacterial Growth in Laboratory Conditions
• Continuous culture • Bacterial culture can be maintained • Continuous exponential growth can be sustained by use of chemostat • Continually drips fresh nutrients in • Releases same amount of waste product 48 49
Environmental Factors on Growth
Environmental Factors on Growth • As group, prokaryotes inhabit nearly all environments • Some live in “comfortable” habitats • Some live in harsh environments • Most of these are termed extremophiles and belong to domain Archaea • Major conditions that influence growth • Temperature • Oxygen • pH • Water availability Environmental Factors on Growth Temperature • Temperature • Psychrophile • Each species has well- • Optimum temperature -5°C to 15°C • Found in Arctic and Antarctic regions defined temperature range • Psychrotroph • Within range lies optimum 20°C to 30°C growth temperature • Important in food spoilage • Mesophile • Prokaryotes divided into 5 25°C to 45°C categories • More common • Disease causing • Thermophiles 45°C to 70°C • Common in hot springs • Hyperthermophiles 70°C to 110°C • Usually members of Archaea • Found in hydrothermal vents Oxygen • Prokaryotes divided based on oxygen requirements • Obligate aerobes • Absolute requirement for oxygen • Use for energy production • Obligate anaerobes • No multiplication in presence of oxygen • May cause death • Facultative anaerobes • Grow better with oxygen • Use fermentation in absence of oxygen • Microaerophiles • Require oxygen in lower concentrations • Higher concentration inhibitory • Aerotolerant anaerobes • Indifferent (neither good nor bad) to oxygen, grow with or without • Do not use oxygen to produce energy Environmental Factors on Growth Environmental Factors on Growth
• pH • Bacteria survive within various pH range • Neutrophiles • Multiply between pH of 5 to 8 • Maintain optimum near neutral • Acidophiles • Thrive at pH below 5.5 • Maintains neutral internal pH, pumping out protons (H+) • Alkalophiles • Grow at pH above 8.5 • Maintain neutral internal pH through sodium ion exchange • Exchange sodium ion for external protons Water • Water availability • All microorganisms require water for growth • Water not available in all environments • In high salt environments • Bacteria increase internal solute concentration • Synthesize small organic molecules • Osmotolerant bacteria tolerate high salt environments • Bacteria that require high salt for cell growth termed halophiles Nutritional Factors on Growth • Growth of prokaryotes depends on nutritional factors as well as physical environment • Main factors to be considered are: • Required elements • Growth factors • Energy sources • Nutritional diversity Nutritional Factors on Growth • Required elements Major elements • Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, magnesium, calcium and iron • Essential components for macromolecules • Organisms classified based on carbon usage • Heterotrophs • Use organism carbon as nutrient source • Autotrophs • Use inorganic carbon (CO2) as carbon source • Trace elements • Cobalt, zinc, copper, molybdenum and manganese • Required in minute amounts Nutritional Factors on Growth • Growth factors • Some bacteria cannot synthesize some cell constituents • These must be added to growth environment • Referred to as growth factors • Organisms can display wide variety of factor requirements • Some need very few while others require many • These termed fastidious Nutritional Factors on Growth
• Energy Sources • Organisms derive energy from sunlight or chemical compounds • Phototrophs • Derive energy from sunlight • Chemotrophs • Derive energy from chemical compounds • Organisms often grouped according to energy source Nutritional Factors on Growth Nutritional Factors on Growth • Nutritional Diversity • Organisms thrive due to their ability to use diverse sources of carbon and energy • Photoautotrophs • Use sunlight and atmospheric carbon (CO2) as carbon source • Called primary producers (Plants) • Chemolithoautotrophs • a.k.a chemoautotrophs or chemolitotrophs • Use inorganic carbon for energy and use CO2 as carbon source • Photoheterotrophs • Energy from sunlight, carbon from organic compounds • Chemoorganoheterotrophs • a.k.a chemoheterotrophs or chemoorganotrophs • Use organic compounds for energy and carbon source • Most common among humans and other animals Laboratory Cultivation • Knowing environmental and nutritional factors makes it possible to cultivate organisms in the laboratory • Organisms are grown on culture media • Media is classified as complex media or chemically defined media • Complex media • Contains a variety of ingredients • There is no exact chemical formula for ingredients • Can be highly variable • Examples include • Nutrient broth • Blood agar • Chocolate agar Laboratory Cultivation • Special types of culture media • Used to detect or isolate particular organisms • Are divided into selective and differential media • Selective media • Inhibit the growth of unwanted organisms • Allow only sought after organisms to grow • Example • Thayer-Martin agar • For isolation of Neisseria gonorrhoeae • MacConkey agar • For isolation of Gram-negative bacteria Laboratory Cultivation • Differential media • Contains substance that bacteria change in recognizable way • Example • Blood agar • Certain bacteria produce hemolysin to break down RBC • Hemolysis • MacConkey agar • Contains pH indicator to identify bacteria that produce acid 66 Laboratory Cultivation • Providing appropriate atmospheric conditions • Bacteria can be grouped by oxygen requirement • Capnophile - Require increased CO2 • Achieve higher CO2 concentrations via • Candle container • CO2 incubator • Microaerophile - Require higher CO2 than capnophile • Incubated in gastight container • Chemical packet generates hydrogen and CO2 gastight container Laboratory Cultivation • Anaerobe • Die in the presence of oxygen • Even if exposed for short period of time • Incubated in anaerobe jar • Chemical reaction converts atmospheric oxygen to water • Organisms must be able to tolerate oxygen for brief period • Reducing agents in media achieve anaerobic environment • Agents react with oxygen to eliminate it • Sodium thioglycollate in media • Anaerobic chamber also used for cultivation 70 Detecting Bacterial Growth • Plate counts • Measures viable cells growing on solid culture media • Count based on assumption that one cell gives rise to one colony • Number of colonies = number of cells in sample • Ideal number to count • Between 30 and 300 colonies • Samples are normally diluted in 10-fold increments • Plate count methods • Pour-plates • Spread-plates methods Detecting Bacterial Growth • Turbidity • Measures with spectrophotometer • Measures light transmitted through sample • Measurement is inversely proportional to cell concentration • Must be used in conjunction with other test once to determine cell numbers • Limitation • Must have high number of cells 73 74
BACTERIAL PATHOGENICITY AND VIRULENCE
• Pathogen • Pathogenicity • Virulence 75
Pathogenicity ◦ Pathogens are organisms that can cause disease in otherwise healthy people That pathogen termed primary pathogen ◦ Microbes that cause disease when the body’s defenses are down termed opportunistic pathogen May be part of normal flora or common in environment ◦ Virulence is quantitative term referring to pathogen’s disease-causing ability Highly virulent organisms have high degree of pathogenicity These organisms more likely to cause disease Example: Streptococcus pyogenes Causes disease from strep throat to necrotizing fasciitis (flesh eating disease 76
