Chapter_2_Detection_Enumeration_Identification_.pdf

DETECTION, ENUMERATION, & IDENTIFICATION Environmental Microbiology Chapter 2 CHAPTER 2 Sample collection & Microscopy Culturing Physio...

DETECTION, ENUMERATION, & IDENTIFICATION Environmental Microbiology Chapter 2 CHAPTER 2 Sample collection & Microscopy Culturing Physiological method processing Immunological method Nucleic-acid based method THE BEGINNING OF ENVIRONMENTAL MICROBIOLOGY Water Quality Initial scientific focus of Environmental Microbiology pathogens Public health WATER QUALITY  Incidence of typhoid fever and cholera are the most frequent cases.  Few treatments were applied to eliminate waterborne bacterial disease.  Filtration  Disinfection by chlorine  In 1960’s, people assuming that threats from waterborne disease had been eliminated.  But, the viruses and protozoa, they are more resistant to general disinfection than enteric bacteria.  Parasite Giardia and Norovirus  Both of which were found in disinfected drinking water 50% OF DIARRHEA-ASSOCIATED ILLNESS IS CAUSED BY WATERBORNE DISEASE QUESTIONS?  How do pathogens survive in the environment?  To know their habitat in order to culture them  How can they be detected and eliminated rapidly and economically to allow both protection of human health and rapid consumption of fresh produce?  Colilert – chlorine – filter  Hence, determining the presence and significance of pathogenic organisms in food, water, and air supplies is a challenge that will keep environmental microbiologists occupied in the coming decades. CIVILIZATION & MODERNIZATION  Human and environmental hygiene efforts have had the greatest impact on reducing human suffering throughout the past century.  Invention of penicillin  This is also true today as we see the evolution of new and more virulent forms of environmentally transmitted pathogens. This has been highlighted by concerns about the spread of SARS-CoV-2 and the potential for the rapid spread of other diseases. NIPAH VIRUS EBOLA VIRUS QUESTIONS?  How do pathogens survive in the environment?  To know their habitat in order to culture them  How can they be detected and eliminated rapidly and economically to allow both protection of human health and rapid consumption of fresh produce?  Hence, determining the presence and significance of pathogenic organisms in food, water, and air supplies is a challenge that will keep environmental microbiologists occupied in the coming decades. SAMPLE COLLECTION & PROCESSING GREAT PLATE COUNT ANOMALY (GPCA)  Discrepancy between the number of microbial cells observed by microscopic examination and the number of colonies that can be cultivated from the same natural sample.  some of the organisms observed upon microscopic examination of natural samples may be truly non-viable.  viable but nonculturable (VBNC)  motility, dividing cells, grows in nature, or stains with dyes for living cells  the right conditions for their growth in the lab have not yet been created.  enrichment cultures 14  Human Oral Microbiome Database lists 707 taxa at the CULTURING TECHNIQUES species level, of which 244 have yet to be cultured  Isolation  The separation of individual organisms from the mixed community 15 CULTURING TECHNIQUES  Selective medium  Allow certain types of organisms to grow  But inhibit the growth of other organisms  Can be achieved by adding dyes, antibiotics, salts or specific inhibitors  MANNITOL SALT AGAR (MSA)  EOSIN METHYLENE BLUE AGAR (EMB agar) CULTURING TECHNIQUES  Enrichment Cultures  Select for desired organisms through manipulation of medium and incubation conditions  originally designed by Martinus Beijerinck, is based upon the use of selective culture media and incubation conditions to isolate targeted microorganisms from natural samples.  selective for a group, or certain groups, of prokaryotes; growth medium components and incubation conditions are chosen to select for a given group and against other groups  nitrogen-fixing bacteria  ENRICHMENT CULTURE FOR NITROGEN-FIXING BACTERIA  Medium with lacking biologically available nitrogen, e.g. Jensen’s nitrogen free agar To isolate N2-fixing bacteria, soil can be added to liquid medium that lack of biologically available nitrogen sources like NH4+ or NO3-. After incubation, N2-fixing bacteria will become 18 dominant in the culture. ENRICHMENT CULTURE FOR NITROGEN-FIXING BACTERIA  Enrichment cultures  When microbes can be grown in the lab setting, they may grow slowly or may be rare in a mixed population.  Enrichment methods promote growth of desired microbes over undesired cells. PERCENTAGE OF CULTURABLE MICROORGANISMS FROM VARIOUS ENVIRONMENTS ▪ Vast majority of microbes have not been grown under laboratory conditions. ▪ Ways of studying microbes that can’t be grown within the context of their natural habitats are important. 20 MICROSCOPY EXAMINATION MICROSCOPY EXAMINATION ASSESSMENT OF MICROBIAL VIABILITY BY USE OF DIRECT STAINING direct staining LIVE/DEAD BacLight Bacterial Viability Living cells stain green, while dead and dying cells stain red, relied on the integrity of cell membrane 23.  FLUORESCENT STAINING Immunofluorescence (IF) is relied on the use of antibodies to label a specific target antigen with a fluorescent dye to visualize the target under fluorescent microscope. MOST PROBABLE NUMBER (MPN) Most probable number (MPN) method for counting coliform 25 MOST PROBABLE NUMBER (MPN)  Technique for estimating the number of microbes in a natural sample  samples include food or water  serial dilutions and observation of growth  microbe must be capable of growth in laboratory  Enumerating bacteria from a natural sample (wastewater, food etc) based upon a ten-fold dilution enrichment strategy of placing an inoculum into a selective culture medium wherein the last tube showing growth, which should have originated from ten or fewer cells, is used as the inoculum for another dilution series.  Pure cultures can be obtained by repeating the process multiple times. The MPN of the selected organism or group of organisms per mL of sample can be determined when using highly selective media and incubation conditions.  Alternatively, an estimation of viable and cultivable organisms can be made using nonselective complex media. MOST PROBABLE NUMBER (MPN)  Traditional methods of enumeration.  But time consuming and labour intensive.  The most probable number (MPN) is a statistical method used to estimate the viable numbers of bacteria in a sample by inoculating broth in 10-fold dilutions and is based on the principle of extinction dilution.  MPN is particularly useful for low concentration of microorganisms (