Name: measuring microbes in samples
Uploaded: 2026-03-12T13:57:41.000Z
Duration: 1 min 30 s
Channel: JoVE

אוכלוסיות מיקרוביאליות הן קבוצות מאותו מין שנמצאות באותה סביבה. הם מוערכים בשיטות שונות.שיטה מבוססת תרביות הנקראת ספירת לוחות ברת קיימא מעריכה תאים חיים על ידי ציפוי דגימות מדוללות על אגר, כאשר כל תא בר קיימא יוצר מושבה נראית לעין.בשיטת המספר הסביר ביותר, דגימות מדוללות בהדרגה מחוסנות ברפליקטים. לאחר מכן, מספר הצינורות המראים גדילה משמש להערכת צפיפות המיקרובים בדגימות.בדיקות ביוכימיות מעריכות פעילות מיקרוביאלית על ידי מעקב אחר תכונות כמו ייצור אנזימים, ומקשרות אוכלוסיות לתפקידים אקולוגיים.לדוגמה, מיקרובים המייצרים תאית ככל הנראה משמשים כמפרקים מרכזיים ביערות.היברידיזציה פלואורסצנטית במקום משתמשת בגלאי פלואורסצנטי הנקשרים לרצפי rRNA ייחודיים למיקרובים ספציפיים.דבר זה מאפשר זיהוי וכימות מיקרוסקופי של טקסונים מיקרוביאליים.ציטומטריית זרימה מנתחת מיקרובים על ידי העברת קרן לייזר אחד אחד.הוא מעריך תכונות כמו גודל התאים, מורכבות ויכולת הקיימות שלהם, ומאפשר כימות בתפוקה גבוהה והערכת תת-אוכלוסייה בקהילות מורכבות.

שיטות להערכת אוכלוסיות מיקרוביאליות

Methods to Assess Microbial Populations

הערכת אוכלוסיות מיקרוביאליות היא קריטית להבנת תפקידי המיקרובים בבריאות, באקולוגיה ובתעשייה. טכניקות משלימות שונות—הן מבוססות תרבית והן מולקולריות—מאפשרות ניתוח מפורט של שפע, גיוון ותפקוד מיקרוביאלי.<h3>ספירת לוחיות ברת קיימא</h3>ספירת הלוחות הקיימת היא שיטה מסורתית מבוססת תרבית המשמשת להערכת מספר המיקרובים החיים בדגימה. לאחר דילול סדרתי, הדגימה נמרחת על פלטות אגר מזינתיות. כל תא בר קיימא יוצר מושבה נראית לעין, המאפשרת ספירה. טכניקה זו נמצאת בשימוש נרחב במיקרוביולוגיה סביבתית, קלינית ומזון, אם כי היא לוכדת רק אורגניזמים ניתנים לגידול, ומייצגת פחות מהמגוון המיקרוביאלי הכולל.<h3>המספר הסביר ביותר (MPN)</h3>המספר הסביר ביותר הוא טכניקה סטטיסטית המשמשת להערכת ריכוז המיקרואורגניזמים החיים בדגימה. הוא כולל הכנת דילול סדרתי וחיסון מספר שכפולים של מדיום גידול. לאחר הדגירה, נוכחות או היעדר גידול מיקרוביאלי מתועדת לאורך הדילולים. תוצאות אלו מנותחות בשיטות סטטיסטיות כדי לחשב את מספר המיקרואורגניזמים הסביר ביותר בדגימה המקורית. MPN שימושי במיוחד לדגימות עם ספירת מיקרוביאליים נמוכה או למיקרובים שקשה לגדל אותם באופן עקבי על מדיה מוצקה.<h3>בדיקות ביוכימיות</h3>בדיקות ביוכימיות מעריכות פעילות מטבולית מיקרוביאלית, ומציעות תובנות לגבי תפקוד המיקרוביה. בדיקות אלו בדרך כלל מודדות את קליטת הסובסטרט, ייצור גז או פעילויות אנזימטיות, כגון קטלז או אוראז. הם חיוניים לזיהוי תפקידים מיקרוביאליים בתהליכים כמו פירוק ומחזור חומרים מזינים ומעקב אחר שינויים בתפקוד הקהילה.<h3>היברידיזציה פלואורסצנטית במקום (FISH)</h3>FISH עושה שימוש בגשושי אוליגונוקלאוטידים מסומנים פלואורסצנטית, אשר נקשרים לרצפי RNA ריבוזומים מסוימים. גשושים אלו מתחברים עם rRNA של מיקרובים מטרה, ומאפשרים את הדמיה ישירה שלהם תחת מיקרוסקופ פלואורסצנטי. FISH מאפשר זיהוי ומיקום מרחבי של טקסונים מיקרוביאליים בקהילות מורכבות, כולל מינים שאינם ניתנים לגידול. עם זאת, הוא מסתמך על מידע רצף ידוע ועל עיצוב מדויק של הגשושים.<h3>ציטומטריית זרימה</h3>ציטומטריית זרימה מנתחת במהירות תאים מיקרוביאליים על ידי העברתם בנפרד דרך קרן לייזר, ומודדת תכונות כמו גודל התא, גרנולריות וחיוניות באמצעות צבעים פלואורסצנטיים. שיטה זו בעלת תפוקה גבוהה מאפשרת אפיון מפורט של אוכלוסיות מיקרוביאליות גדולות. בשילוב עם סמנים פלואורסצנטיים ממוקדים, הוא יכול להבדיל בין תת-אוכלוסיות או מצבים פיזיולוגיים. עם זאת, הבחנה בין מינים קרובים עשויה לדרוש ספציפיות נוספת באמצעות נוגדנים מסומנים או גלאים גנטיים.

Assessing microbial populations is crucial for understanding microbial roles in health, ecology, and industry. Various complementary techniques&#8212;both ...

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Education

JoVE Core

Microbiology

מיקרוביולוגיה

Unit

יחידה

Microbial Ecology

אקולוגיה מיקרוביאלית

KEY TERMS AND CONCEPTS

מונחים ומושגים מרכזיים

measuring microbes in samples

Measuring microbes in a sample helps scientists study health, ecology, and industry. Microbial counts can show how many cells are present, how diverse they are, and what they may be doing. To do this, researchers use both culture-based and molecular methods.

A viable plate count is a classic culture method for estimating living microbes. The sample is first diluted step by step, then spread on nutrient agar plates. Each viable cell can grow into a visible colony, so the colonies can be counted. This method is useful in environmental, clinical, and food microbiology, but it only detects microbes that can grow in culture. As a result, it can miss part of the total microbial diversity.

The Most Probable Number, or MPN, is a statistical way to estimate the amount of viable microorganisms. It also uses serial dilutions, but the dilutions are placed into several replicate tubes or wells of growth medium. After incubation, scientists record where growth does or does not appear. They then use statistics to calculate the most likely number of microbes in the original sample. MPN is especially helpful when microbial numbers are low or when microbes do not grow reliably on solid media.

Biochemical tests look at microbial metabolism, which means how microbes use chemicals to carry out life processes. These tests can measure substrate uptake, gas production, or enzyme activity such as catalase or urease. They help reveal microbial function and can support identification of roles in decomposition and nutrient cycling. They also help track changes in community function.

Fluorescence in situ hybridization, or FISH, uses fluorescent probes that bind to specific ribosomal RNA sequences. The probes attach to the rRNA of target microbes, so the cells can be seen directly under a fluorescence microscope. This method can identify and localize microbes in complex communities, including species that cannot be cultured. It depends on known sequence information and careful probe design.

Flow cytometry is a fast method for analyzing microbial cells one by one. Cells pass through a laser beam, and the instrument measures features such as size, granularity, and viability with fluorescent dyes. This high-throughput approach can characterize large populations and, with targeted fluorescent markers, can separate subpopulations or different physiological states. However, closely related species may still need extra specificity from labeled antibodies or genetic probes.

Measuring Microbes in Samples

introduction to microbial ecology

Microbial Communities in Soil and Extremes

Microbial ecology looks at how microorganisms live, interact, and adapt in different ecosystems. It also shows how these tiny organisms help shape nutrient cycling, energy flow, and environmental balance. An ecosystem includes living parts and non-living parts, and microbes respond to both. Microorganisms can form populations, which are groups of the same species living together at the same time. When populations from different species live and interact in the same place, they form a microbial...

ecological niche

Microbial Niches in Ecosystems

Microbial niches describe how microorganisms survive, grow, and interact in an ecosystem. A niche includes how a microbe gets resources, reproduces, and relates to other species in its environment. This idea helps explain microbial community dynamics, biogeography, and ecosystem function. A microorganism’s fundamental niche is the full range of conditions where it could survive, grow, and reproduce. These conditions include both biotic factors, such as living interactions, and abiotic factors,...

methods to assess microbial populations

Measuring microbes in a sample helps scientists study health, ecology, and industry. Microbial counts can show how many cells are present, how diverse they are, and what they may be doing. To do this, researchers use both culture-based and molecular methods. A viable plate count is a classic culture method for estimating living microbes. The sample is first diluted step by step, then spread on nutrient agar plates. Each viable cell can grow into a visible colony, so the colonies can be counted.

methods to assess microbial communities

Measuring Microbial Diversity and Activity

Microbial communities include bacteria, archaea, and eukaryotic microorganisms. They live in many ecosystems and affect important environmental and biological processes. Scientists study these communities by looking at how many species are present, how many individuals each species has, and how evenly the species are spread out. Species richness is the number of different species in a sample. Species abundance is the relative amount of each species. Species evenness describes how uniformly the...

the winogradsky column

Microbial Layers Inside a Winogradsky Column

A Winogradsky column shows how microbial layers form in a simple, sealed ecosystem. It is a clear model for studying microbial ecology and how different metabolisms interact in sediment over time. The setup uses a transparent glass cylinder. Half of it is filled with sediment mixed with cellulose, such as shredded paper, calcium carbonate, and gypsum. The cellulose provides a carbon source. Calcium carbonate buffers pH, and gypsum supplies sulfate. The rest of the cylinder is filled with pond...

microenvironments

Microbial Niches in Tiny Habitats

Microorganisms live in tiny habitats called microenvironments. These spaces have their own physical and chemical conditions. Oxygen level, pH, temperature, light, and nutrient supply can all differ from the surrounding area. These local conditions strongly affect microbial growth, metabolism, and which species can live there. Microenvironments often have sharp gradients over very small distances. In soil, oxygen may be available at the particle surface but drop off just a few millimeters below.

microbial mats

Microbial Mats and Oxygen Gradients

Microbial mats are layered communities of microbes that grow in response to changes in light, oxygen, and nutrients. These ecosystems are highly organized, and different microorganisms live at different depths. Biofilms are the early stage of this growth and are only a few millimeters thick. Mature microbial mats can grow to centimeter scale and show clear vertical structure. This layered arrangement lets microbes fill separate niches within a very small space. In cyanobacterial mats from hot...

microbial interactions: mutualism

Mutualistic Partnerships in Nature

Mutualistic partnerships in nature show how two or more organisms can benefit from living together. These symbiotic relationships can be obligate, meaning the partners depend on each other, or facultative, meaning the association is helpful but not required. Mutualism helps support many ecosystem functions across land and water. One common example is the partnership between plant roots and mycorrhizal fungi, such as Rhizophagus species. The fungal hyphae grow into the root hairs and epidermis,...

microbial interactions: cooperation

Microbial Cooperation in Soil and Hosts

Microbial cooperation describes helpful interactions between different species. In these partnerships, microbes work together for one or mutual benefit. These interactions can shape ecosystems and evolution. They also play major roles in symbiosis and disease. One clear example involves the Gram-negative bacterium Xenorhabdus nematophila and the parasitic nematode Steinernema carpocapsae. Juvenile nematodes carry the bacteria in the soil and need an insect host to mature. When the nematode...

microbial interactions: predation

How Microbes Hunt and Defend

Microbial predation is a form of interaction in which one microorganism kills and eats another to gain nutrients and energy. It includes both bacterial predators and protozoan predators. This process helps shape microbial communities and affects how nutrients move through the environment. Some bacterial predators attack from the outside, while others enter the prey cell. Epibiotic predators, such as Vampirococcus, attach to the surface of a prey cell and release hydrolytic enzymes. These...

microbial interactions: competition

Microbial Competition and Niche Partitioning

Microbial competition shapes how bacteria and other microbes share space and resources. In shared environments, they compete for nutrients, space, or light. The strength of this competition depends on the environment, including nutrient levels, space limits, and how many microbial species are present. These interactions help determine the structure, function, and resilience of microbial communities. Microbial competition is usually grouped into exploitative competition and interference...

microbial interactions: parasitism

Microbial Parasites and Host Dependence

Microbial parasites depend on a host for nutrients and shelter, and this dependence often harms the host. In parasitism, only the parasite benefits. This is different from mutualism, where both organisms gain from the relationship. Microbial parasites are classified by where they live in or on the host. Ectoparasites stay on the host’s surface, such as the skin or outer tissues, and take in nutrients from outside. Endoparasites live inside the host’s body and invade tissues or organs.

microbe-plant interactions

How Plant Microbes Affect Growth

Plant microbes can help, harm, or simply live alongside their hosts. These microbe-plant interactions shape plant growth, health, and ecosystem function. The plant microbiome includes bacteria, fungi, archaea, protists, and viruses, and these organisms may live on the plant surface, inside plant tissues, or in symbiosis throughout the plant. Some of the most important beneficial partnerships involve mycorrhizal fungi. These fungi form close connections with roots and are grouped as arbuscular...

microbes and the carbon cycle

How Microbes Move Carbon Through Earth

Microbes move carbon through Earth’s major reservoirs and help drive the carbon cycle. Carbon shifts among the biosphere, lithosphere, atmosphere, and hydrosphere, where it supports life and helps regulate climate. It commonly cycles as carbon dioxide (CO2), the oxidized form of carbon, but methane (CH4) and organic compounds also matter. Photosynthetic microbes take in atmospheric CO2 and turn it into organic carbon. Cyanobacteria are especially important because they carry out oxygenic...

microbes and methanogenesis

How Microbes Produce Methane

Methane production in anaerobic ecosystems depends on methanogenic archaea and the microbes that work with them. This process happens where oxygen is absent, such as wetlands, sediments, and animal gastrointestinal tracts. In these anoxic habitats, aerobic respiration cannot run, so microbes use other pathways to break down organic matter. Anaerobic decomposition starts when cellulolytic bacteria, or cellulose-digesting bacteria, hydrolyze dead organic material into simple sugars. Other...

microbes and the nitrogen cycle

Microbial Steps in the Nitrogen Cycle

Microbes drive the nitrogen cycle by changing nitrogen into forms that plants and other organisms can use. This biogeochemical cycle keeps nitrogenous compounds balanced in ecosystems. It also supports plant growth and microbial growth. Nitrogen fixation begins the cycle by converting inert atmospheric nitrogen gas, N2, into ammonia, NH3. This step depends on nitrogenase enzymes. Free-living diazotrophs do this on their own. Aerobic bacteria such as Azotobacter spp. and anaerobic bacteria such...

microbes and the sulfur cycle

Sulfur Cycling Through Microbial Reactions

Sulfur cycling depends on microbial reactions that move sulfur through rocks, water, soil, and the air. In Earth systems, sulfur shifts among inorganic forms such as sulfate, elemental sulfur, and sulfide. These changes happen through both abiotic processes and biological activity in oxic and anoxic environments. Sulfate is the most oxidized form of sulfur. It is stored mainly in rocks, marine sediments, and ocean water, where it acts as a long-term reservoir in the global sulfur cycle. In...

microbes and other elemental cycles

Microbes Driving Iron and Manganese Cycles

Microbes drive iron and manganese cycling in aquatic environments by changing these metals between reduced and oxidized forms. These reactions are especially important along redox gradients, where oxygen levels shift across stratified water layers. The microbes use these metals for energy, growth, and structural needs. In oxygen-rich surface waters, iron is usually present as insoluble ferric iron, Fe(III). Some bacteria release siderophores, which are small organic molecules that bind ferric...

marine microbial ecology

Microbial Life in the Ocean

Marine microbial life is shaped by salinity, low nutrients, and changing oxygen levels. These physical and chemical limits influence which microbes can survive and how they function. They also affect how ocean microbes help drive nutrient and carbon cycling. Small cell size is common in these environments because it gives microbes a larger surface-to-volume ratio. That helps them take up nutrients more efficiently when nutrients are scarce. In estuaries, where salinity changes often,...

deep sea microbial ecology

Deep Sea Microbes and Extreme Conditions

Deep sea microbes live in one of Earth’s harshest environments. The deep ocean is far below the sunlit photic zone, which usually reaches the upper 100 to 200 meters of open water. Below that layer, the bathypelagic and abyssal zones stretch from about 1,000 to 6,000 meters, and hadal trenches go even deeper. This region is dark, cold, and highly pressurized. Temperatures are usually around 2 to 3°C, although hydrothermal vents can be slightly warmer. Pressure rises by about 1 atmosphere for...

freshwater microbial ecology

Microbes in Streams, Lakes, and Rivers

Freshwater microbes live in streams, rivers, lakes, ponds, and marshes, but their communities change with water movement, light, depth, and nutrients. These systems are often grouped into lotic waters, which flow, and lentic waters, which are still or slow-moving. The physical setting shapes where microbes grow and how they get energy. In lentic systems, phytoplankton are the main producers. They make autochthonous organic carbon, which means carbon made within the water body. In lotic...

soil microbial ecology

How Soil Microbes Cycle Nutrients

Soil microbes drive nutrient cycling, organic matter turnover, and ecosystem stability. A gram of soil can contain thousands of bacterial and archaeal taxa, but their shared job is what helps sustain land ecosystems. Their activity changes the way carbon, nitrogen, and other nutrients move through soil. Soil is a patchwork of minerals, organic matter, water, and air. Microbes live in tiny microhabitats shaped by pore size, moisture, oxygen, and nutrients. Sandy soils drain fast and tend to...

key terms and concepts

Explore how scientists measure microbial populations with plate counts, MPN, biochemical tests, FISH, and flow cytometry in health, ecology, and industry.

Measuring Microbes in Samples | JoVE K12

JoVE Core Microbiology provides an engaging introduction to the microbial world and its influence on health, industry, and the environment. This comprehensive collection covers key concepts such as microbial structure, metabolism, genetics, and ecology. It introduces essential laboratory techniques and microscopy methods through visually engaging animations. Corresponding scientist-in-action videos bridge classroom concepts with real-world research and applications.

microbiology

Measuring Microbes in Samples

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