Bacterial Growth and Diagnosis PDF

Bacterial growth and diagnosis 1 Asst. Prof. Ayhan Mehmetoğlu  Learning objectives Student should know growth requirements of bacteria Stages of the bacterial growth cycle Overview of metabolism Aerobic respiration, anaerobic res...

Bacterial growth and diagnosis 1 Asst. Prof. Ayhan Mehmetoğlu  Learning objectives Student should know growth requirements of bacteria Stages of the bacterial growth cycle Overview of metabolism Aerobic respiration, anaerobic respiration and fermentation Diagnosis of bacterial infection 2  Physical Requirements The Requirements for Bacterial Growth Chemical Requirements  Temperature Physical pH Requirements Osmotic Pressure  Carbon Nitrogen Chemical Phosphorus Requirements Oxygen Hydrogen CHONPS Trace Elements  A distinctive feature of bacterial metabolism is the variety of mechanisms used to generate energy from these carbon sources. Energy production According to the biochemical mechanism used, bacterial metabolism can be categorized into three types: Anaerobic respiration, Aerobic respiration and fermentation 6  Simply, respiration, is the metabolic process in which molecular oxygen serves as the final electron acceptor of the electron transport chain. Aerobic In this process, oxygen is respiration reduced to water. Respiration is the energy- generating mode used by all aerobic bacteria. 7  Inorganic compounds other than molecular oxygen serve as the final electron acceptors. Anaerobic Acceptors can be molecules such as nitrate or sulfate. respiration Anaerobic respiration can be used as an alternative to aerobic respiration in some species (facultative organisms), 8 9  All cells must accomplish certain metabolic tasks to maintain life. All cells, whether bacterial or human, accomplish these metabolic tasks by Growth and similar pathways. Metabolism There are, however, some important differences that set bacteria apart metabolically from eukaryotic cells, and these differences can often be exploited in the development of 10 antibacterial therapies.  Characteristics of bacterial growth If bacterial cells are suspended in a liquid nutrient medium, the increase in cell number or mass can be measured in several ways. Microscopically Counting the number cells that are able to form colonies in a solid nutrient (agar) Quantitating the turbidity 11 Stages of Because bacteria reproduce by binary fission, with time the (the exponential or log phase of growth). bacterial growth Depending on the species, the minimum doubling cycle time can be as short as twenty minutes or as long as several days. 12  Single E. coli ten million cells in just eight hours. Stages of Eventually, growth slows and ceases entirely (stationary the phase) as nutrients are depleted bacterial and toxic waste products accumulate. growth Most cells in a stationary phase cycle are not dead, however. If they are diluted into fresh growth medium, exponential growth will resume after a lag phase. 13 Lippincotts_Illustrated_Reviews_Microbiology_3rd_Edition_by_Richard_A._Harvey_Cynthia_Nau_Cornelissen_Ph.D 14  Stages of the bacterial growth cycle 1. The first is the lag phase, during which vigorous metabolic activity occurs but cells do not divide. This can last for a few minutes up to many hours. 2. The log (logarithmic) phase is when rapid cell division occurs. Note: Beta-Lactam drugs, such as penicillin, act during this phase because the drugs are effective when cells are making peptidoglycan, i.e., when they are dividing. 15  Stages of the bacterial growth cycle 3. The stationary phase occurs when nutrient depletion or toxic products cause growth to slow until the number of new cells produced balances the number of cells that die resulting in a steady state. 4. The final phase is the death phase, which is marked by a decline in the number of viable bacteria. 16  If a single bacterial cell is placed on a solid nutrient agar surface it can form a compact macroscopic mass of cells (colony). For rapidly growing species, overnight Surface incubation at 30° to 37°C is sufficient to produce visible colonies, each containing growth millions of cells. The gross characteristics of colonies (for example, color, shape, adherence, smell, and surface texture) can be useful guides for identification of the species of bacterium.17 18 Growth of bacterial colonies Growth of bacterial colonies on a solid, nutrient surface, for example, nutrient agar. [Note: The doubling time of bacteria is assumed to be 0.5 hr.] 19  Culture Medium: Nutrients prepared for microbial growth Culture Sterile: No living microbes Media Inoculum: Introduction of microbes into medium Culture: Microbes growing in/on culture medium  Complex polysaccharide Used as solidifying agent for culture media in Petri plates, slants, and deeps Agar Generally not metabolized by microbes Liquefies at 100°C Solidifies ~40°C  Anaerobic Culture Methods Reducing media Heated to drive off O2 Contain chemicals that combine O2 Anaerobic jar Anaerobic chamber Candle jar and CO2-packet  Selective Media: Suppress unwanted microbes and encourage desired microbes. Types of Differential Media: culture Make it easy to distinguish colonies of media different microbes. Enrichment Media: Encourages growth of desired microbe Petri dish Slant and deep media Examples of BLOOD AGAR MACCONKEY AGAR selective media HEKTOEN MANNITOL SALT ENTERIC AGAR AGAR (MSA) (HE)  Chocolate agar Is a non-selective, enriched growth medium Contains red blood cells, which have been lysed by heating  Lowenstein- Jensen Medium For the cultivation and differentiation of Mycobacterium species Incubate: 3 days Mueller-Hinton agar (MHA) For antimicrobial susceptibility testing 34 Dr. Ayham Abulaila 35 Introduction to diagnostic microbiology Diagnosis of Bacterial Infection Patient Clinical Non-microbiological diagnosis investigations Radiology Haematology Biochemistry Sample Take the correct specimen Take the specimen correctly Label & package the specimen up correctly Appropriate transport & storage of specimen  Problems in delay or inappropriate storage delay in diagnosis & treatment pathogens die Getting the contaminants overgrow specimen Blood cultures directly into incubator not refrigerator! to the lab CSF straight to lab Don't put an entire surgical specimen into formalin! Send a portion to microbiology in a sterile container  Take an mid-stream urine avoids contamination with perineal flora Collecting CSF the Avoid contamination Avoid bloody tap specimen Throat swab correctly Make the patient gag! Blood cultures Avoid contamination with skin organisms  wrong sample e.g. saliva instead of sputum Factors delay in transport / inappropriate storage limiting e.g. CSF usefulness of overgrowth by contaminants bacteriological e.g. blood cultures investigations insufficient sample / sampling error e.g.in mycobacterial disease patient has received antibiotics  Diagnosis of Bacterial Infection microscopy unstained or stained with e.g. Gram stain Stain Decolorise Counterstain culture identification by biochemical or serological tests on pure growth from single colony on plates or in broth sensitivities by disc diffusion methods, breakpoints or MICs Serodiagnosis DNA technologies 24 ،‫ تشرين األول‬24  Microscopy Unstained preparations “Wet prep” Dark-ground illumination for syphilis  Microscopy Stained preparations Gram-stain Acid-fast stain Ziehl-Neelsen Fluorescence Direct, e.g. auramine Immunofluorescence  Culture of Bacteria Solid media: Agar plates For Identification For Enumeration Slopes/ For safe long-term culture, e.g. Lowenstein-Jensen media for TB Liquid media (broth): For enrichment or maximum sensitivity  Identification of Bacteria Morphology Growth requirements Biochemistry Enzymes Antigens  Bacteria reproduce by binary fission, whereas eukaryotic cells reproduce by mitosis. The bacterial growth cycle consists of four phases: the lag phase, during which nutrients are incorporated; the Summary log phase, during which rapid cell division occurs; the stationary phase, during which as many cells are dying as are being formed; and the death phase, during which most of the cells are dying because nutrients have been exhausted. 46  Some bacteria can grow in the presence of oxygen (aerobes and facultatives), but others die in the presence of oxygen (anaerobes). The use of oxygen by bacteria generates toxic products such as superoxide and Summary hydrogen peroxide. Aerobes and facultatives have enzymes, such as superoxide dismutase and catalase, that detoxify these products, but anaerobes do not and are killed in the presence of oxygen. 47 ‫‪ 24‬تشرين األول‪24 ،‬‬  Refernces Murray, Patrick R., MEDICAL MICROBIOLOGY, EIGHTH EDITION ISBN: 978-0-323-29956-5 Lippincotts Illustrated Reviews Microbiology 3rd Edition by Richard A. Harvey Cynthia Nau Cornelissen_Ph.D Jawetz Melnick & Adelbergs Medical Microbiology Review of Medical Microbiology and Immunology, Fourteenth Edition by Warren Levinson, MD, PhD. www.cdc.com For any question [email protected] 50