Pharm 214L Unit 2 Handout - STUDENT COPY PDF

UNIT 2 CULTURE MEDIA, INOCULATION & INCUBATION TECHNIQUES Microbiology is the study of microorganisms such as bacteria, protozoa, fungi and similar organisms that At the end of this lesson, you should be able to: can’t be seen with the...

UNIT 2 CULTURE MEDIA, INOCULATION & INCUBATION TECHNIQUES Microbiology is the study of microorganisms such as bacteria, protozoa, fungi and similar organisms that At the end of this lesson, you should be able to: can’t be seen with the naked eye. The need to study Identify the various types of culture media these minute organisms started when scientists Know the various methods to maintain the discovered the association of microbes to specific required environmental conditions during diseases. The roles of microbiology on the advances in incubation the healthcare industry, especially in pharmaceutical Demonstrate proper incubation and medical industry have led to great discoveries, from techniques of bacterial cultures vaccines to devices. Understanding microbial growth and its similarities and differences with human cell mechanisms allows pharmacists to discover antimicrobial drugs that would prevent an escalating number of communicable diseases and control the growth of pathogenic microorganisms. I. DEFINITION OF TER MS 1. Microbial Growth: increase in cell number through binary fission 2. Binary Fission: cell simply splits into two cells, producing two identical cells Generation Time: time required for a cell to divide, and its population to double. 3. Budding: cells form an initial outgrowth (a bud) that enlarges its size until it approaches that of a parent cell, and then it separates 4. Culture: microbes that grow and multiply in or on a culture medium 5. Colony: a group of descendants of an original cell 6. Pure Culture: aka axenic; contains a single microbial species II. PHYSICAL REQUIREMENTS FOR GROWTH A. TEMPERATURE PSYCHROPHILES PSYCHROTROPHS MESOPHILES THERMOPHILES − sensitive to − optimum growth at − optimum growth at − optimum growth at temperatures over 20˚C to 30˚C 25˚C to 40˚C 50˚C to 60˚C 20˚C − Extreme − optimum growth at Thermophiles 15˚C or below grow at 80˚C or higher Pharm 214L Faculty © 2024 Saint Louis University | Department of Pharmacy 1 Acknowledgments to: Ma'am Jill Marie G. Arabia  B. pH ACIDOPHILES NEUTROPHILES ALKALIPHILES − grow at very low pH (0.1 to − grow at pH 5.4 to 8.5 − grow at high pH (7 to 12) 5.4) − Vibrio cholerae − Lactobacillus acidophilus C. OSMOTIC PRESSURE OBLIGATE HALOPHILES FACULTATIVE HALOPHILES − require very high salt conc. 20 to 30% − do not require high salt conc. for growth − aka extreme halophiles − tolerates 2-15% salt concentration III. CHEMICAL REQUIREMENTS FOR GROWTH A. CARBON CHEMOHETEROTROPHS CHEMOAUTOTROPHS − organisms which derive energy from organic − organisms which use inorganic energy sources to carbon sources synthesize organic compounds − may need to consume other living organisms to − primary carbon source is carbon dioxide live B. NITROGEN C. SULFUR D. PHOSPHORUS E. OTHERS − makes up 14% of − used to form − used to form DNA, − K dry cell weight proteins and some RNA, ATP, and − Mg vitamins phospholipids − Ca − Sources: proteins, − Fe ammonium, − Sources: proteins, − Sources: Inorganic − Cu nitrogen gas, hydrogen sulfide, phosphate salts, − Mo nitrates sulfates Buffers (from − Zn culture medium) Pharm 214L Faculty © 2024 Saint Louis University | Department of Pharmacy 2  F. OXYGEN − Growth requirement for aerobic microorganisms Sources: Atmosphere, Water OBLIGATE FACULTATIVE OBLIGATE AEROTOLERANT MICRO- AEROBES ANAEROBES ANAEROBES ANAEROBES AEROPHILES − Require − Can use − Can’t use − Can’t use − Require oxygen oxygen to live oxygen, but oxygen and oxygen, but at low can grow in its are harmed can grow in its concentrations − Mycobacterium absence by the absence tuberculosis presence of − Helicobacter − Escherichia coli toxic forms of − Tolerates pylori oxygen presence of oxygen − Clostridium tetani − Streptococcus pneumoniae OXYGEN IN ANAEROBES Toxic Bacterial O2 Metabolism Products Death O2- OH- O2+ H2O2 OXYGEN IN AEROBES Toxic Detox Non-toxic O2 Metabolism Products Pathways SOD, Catalase, Products O2- OH- O2+ H2O2 H2O O2 Peroxidase ENZYMES REQUIRED TO NEUTRALIZE TOXIC FORMS OF O 2 Superoxide free radical 1. Superoxide dismutase: converts superoxide to oxygen & hydrogen peroxide O2- + O2- + 2H+ → H2O2 + O2 Peroxide anion 1. Catalase: converts hydrogen peroxide to water & oxygen 2 H2O2 → 2H2O + O2 2. Peroxidase: breaks down hydrogen peroxide H2O2 + 2H+ → 2H2O Pharm 214L Faculty © 2024 Saint Louis University | Department of Pharmacy 3  G. CARBON DIOXIDE CAPNOPHILES − CO2-loving − require increased concentration of CO2 and lower levels of O2 IV. BACTERIAL GROWTH CURVE Pharm 214L Faculty © 2024 Saint Louis University | Department of Pharmacy 4 V. CULTURE MEDIA − substance containing nutrients in which cells or microorganisms can be grown; may be solid, liquid or gel − Characteristics: i. Contains appropriate nutrients ii. Sterile iii. Incubated at appropriate temperature − Inoculation: act of introducing microorganism or suspension of microorganisms into a culture medium − Inoculum: microbial cells added to a medium in order to start a culture A. SOLID M EDIA: nutrient material that contains a solidifying agent 1. Agar Plate − Allows for isolation and examination of colonies − Difficult to transport and store 2. Agar Deep − Inoculating needle is used to “stab” the solid medium − For microorganisms that prefer reduced O2 3. Agar Slant − Tube of solid medium at an angle for higher surface area − For easy storage and transportation AGAR − Unbranched polysaccharide obtained from the cell walls of some species of red algae such as: i. Gracilaria compressa ii. Gelidium cartilagineum iii. Gelidium amansii − Melts at above 95˚C; once melted, does not solidify until it reaches 40˚C − Cannot be degraded by most bacteria B. LIQU ID M EDIA: nutrient material that lacks a solidifying agent 1. Broth − Liquid medium in a tube, bottle, or flask − Applicable if high concentration of bacteria is desired Growth of Liquid Medium Depending on Oxygen Requirements C. SEM ISOL ID ME DIA : nutrient material that contains