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Environmental Microbiology
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JoVE Science Education Environmental Microbiology
Culturing and Enumerating Bacteria from Soil Samples
  • 00:00Overview
  • 01:25Principles of Culturing and Enumerating Soil Bacteria
  • 03:33Preparation of Soil Dilutions
  • 04:29Making Spread Plates for Bacterial Culture
  • 06:20Bacterial and Actinomycetes Counts
  • 07:09Isolation of Pure Cultures
  • 08:41Applications
  • 10:31Summary

培养和枚举从土壤样品的细菌

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Overview

资料来源: 实验室的博士伊恩胡椒和博士查尔斯称-亚利桑那大学
演示作者: 布拉德利施密茨和路易莎 Ikner

表层土壤是的拼在一起就形成二次聚合的无机和有机颗粒均匀的混合物。内和骨料之间的空隙或视觉上包含两个的毛孔空气和水。这些条件创建一个理想的生态系统为细菌,所以所有土壤都包含了数量庞大的细菌,通常超过 100 万每克土壤。

细菌是最简单的微生物,称为原核生物。在原核该组内有丝状微生物称为放线菌。放线菌是实际上的细菌,但他们经常被认为是一个独特的群体内的细菌分类由于其丝状的结构,组成的多个单元格,向窗体菌丝串在一起。本实验使用甘油案例媒体选择为放线菌的殖民地,在稀释和电镀。通常情况下,放线菌的细菌总人口的大约 10%。细菌和放线菌发现在每个环境中在地球上,但这些微生物在土壤中的多样性和丰富是无与伦比。这些微生物,对人类生活和什么人吃、 喝、 呼吸,或触摸的影响至关重要。此外,还有细菌物种可以感染人并导致疾病,还有细菌能产生能治病的天然产品。放线菌是产生抗生素,如链霉素的尤为重要。细菌是养分循环、 植物生长和有机污染物的降解的关键。

细菌是物种的高度多样化可以找到在土壤中,部分因为他们生理和代谢功能多样数目。细菌可以是异养,意思他们利用有机的化合物,如葡萄糖、 食品和能源,或自养,意思他们利用无机化合物,如元素硫,对食品和能源。它们也可以是有氧,利用氧气呼吸,或厌氧,利用相结合形式的氧气,如硝酸或硫酸,能够自由呼吸。某些细菌可以使用氧气或组合形式的氧和被称为兼性厌氧菌。

Principles

Procedure

1.土壤稀释液的制备 开始执行程序,称出 10 克的土壤样本,并将添加到 95 mL 的去离子水。摇匀,暂停和”A”的标签。 土壤落定之前,删除 1 mL 悬浮液与不育吸管和移交 9 mL 去离子的水的空白。涡彻底,和作为”B”的标签。 重复此稀释步骤三次,每次 1 毫升的前悬架和 9 毫升与去离子水空白。标签这些顺序作为管 C、 D 和 e。这将导致 10-1 10-5克每毫升土?…

Results

A 10-g sample of soil with a moisture content of 20% on a dry weight basis is analyzed for viable culturable bacteria via dilution and plating techniques. The dilutions were made as shown in Table 1. 1 mL of solution E is pour-plated onto an appropriate medium and results in 200 bacterial colonies.

Equation 1

But, for 10 g of moist soil,

Equation 2

Therefore,

Equation 3

Step Dilution
10 g soil (weight/volume) 95 mL saline (solution A) 10-1
1 mL solution A (volume/volume) 9 mL saline (solution B) 10-2
1 mL solution B (volume/volume) 9 mL saline (solution C) 10-3
1 mL solution C (volume/volume) 9 mL saline (solution D) 10-4
1 mL solution D (volume/volume) 9 mL saline (solution E) 10-5

Table 1: Dilution and plating of the samples.

Applications and Summary

There are two fundamental applications of dilution and plating of soil bacteria. The first application is the enumeration of culturable bacteria within a particular soil. The quantification of the number of soil bacteria gives an indication of soil health. For example, if there are 106 to 108 culturable bacteria present per gram of soil, this would be considered a healthy number. A number less than 106 per gram indicates poorer soil health, which may be due to a lack of nutrients as found in low organic matter soils; abiotic stress imposed by extreme soil pH values (pH < 5 or > 8); or toxicity imposed by organic or inorganic anthropogenic contaminants.

The second major application is the visualization and isolation of pure cultures of bacteria. The pure cultures can subsequently be characterized and evaluated for specific characteristics that may be useful in either medical or environmental applications. Examples include: antibiotic production; biodegradation of toxic organics; or specific rhizobia useful for nitrogen fixation by leguminous crops, such as peas or beans.

Transcript

Determining an accurate count of environmental bacteria is critical to assessing the health of a soil ecosystem. This can be accomplished by culturing bacterial colonies with appropriate dilutions.

Soil bacteria are an important part of a healthy soil ecosystem, playing roles in decomposition, nitrogen fixing, and nutrient cycling. Surface soils are a substrate of organic and inorganic particles forming a complex matrix surrounding pores that fill with air or water. These conditions create an ideal ecosystem for bacteria, with surface soils typically containing upwards of a million bacteria per gram.

Because of this high concentration of bacteria, dilution is necessary before plating onto growth media. Serial dilutions can reduce the concentration of the original soil sample to levels low enough for single colonies to be grown on media plates, allowing for the calculation of the initial counts of bacteria in the soil sample.

This video will illustrate how to prepare serial dilutions of soil samples, how to plate these bacterial samples, and how to calculate soil bacterial counts from the dilution plates.

Bacteria are simple prokaryotic organisms, but highly diverse in terms of species and ecosystem. Actinomycetes are a subset of bacteria that are frequently considered a unique group due to their filamentous structure, where they grow strung together to form hyphae.

Typically, actinomycetes account for 10% of the total bacterial population in soil. Bacteria and actinomycetes are abundant in almost every environment on Earth, but are found in unparalleled diversity and abundance in soil.

Soil bacteria are enumerated, and potentially cultured and identified by dilution plating. Here, a soil sample is serially diluted in water, and then dispersed onto agar growth plates. The resulting colonies are then counted. This value, along with the dilution factor, is used to elucidate the initial concentration of bacteria in soil.

Dilution plating is a common, inexpensive, and simple method for bacterial enumeration, but it does suffer from several drawbacks. The soil should not be allowed to settle during dilutions, which could lead to biased growth. Some soil bacteria will not culture on the plates, or grow too slow to be observed. Additionally, this method assumes that colonies are formed from a single bacterium, but they may arise from clumps of multiple cells.

Certain bacterial species grow better on different growth media. A common substrate to culture a wide range of bacteria is an agar-peptone yeast plate. Actinomycetes preferentially grow on glycerol-casein based plates, which better suit the growth of these filamentous bacteria.

Now that you understand the concept behind bacterial enumeration, let’s see how this process is carried out in the laboratory.

To begin the procedure, weigh out 10 g of soil sample, and add to 95 mL of deionized water. Shake the suspension well, and label as “A”. Before the soil settles, remove 1 mL of the suspension with a sterile pipette and transfer it to 9 mL of deionized water. Vortex thoroughly, and label as “B”.

Repeat this dilution step 3 times, each time with 1 mL of the previous suspension and 9 mL of deionized water. Label these sequentially as tubes C, D, and E. This results in serial dilutions of 10-1 through 10-5 grams of soil per mL.

To grow bacterial colonies, take 3 pre-prepared peptone-yeast agar plates and label them as C, D, and E. Vortex the corresponding samples and pipette 0.1 mL onto each plate. Since CFUs are reported per mL, using 0.1 mL further increases the dilution by a factor of ten.

Next, dip a glass spreader into ethanol. Place the spreader in a flame for a few seconds to ignite and burn off the ethanol. This will sterilize the spreader.

Hold the spreader above the first plate until the flame is extinguished. Open the plate quickly, holding the lid close by. Touch the spreader to the agar away from the inoculum to cool, and then spread the drop of inoculum around the surface until traces of free liquid disappear. Replace the plate lid.

Re-flame the spreader and repeat the process with the next plate, working quickly so as not to contaminate the plate with airborne organisms. Invert the plates to prevent moisture drops falling onto the agar, and incubate the plates at room temperature for one week.

To grow actinomycetes, take three pre-prepared glycerol-casein plates and label them as B, C, and D. Using the techniques shown previously, spread plate 0.1 mL from the suspensions B, C, and D. The lower dilutions are used because actinomycetes are typically present as 1/10th of the bacterial population.

Finally, invert these plates and store at room temperature for two weeks.

After incubation, examine all of the bacteria plates carefully, and note differences in colony size and shape. When grown on agar, bacteria produce slimy colonies ranging from colorless to bright orange, yellow, or pink. In contrast, actinomycete colonies are chalky, firm, leathery, and will break under pressure, where other bacterial colonies will smear. This allows colonies to be distinguished by touch with a sterile loop.

Count and record the number of bacterial colonies, including any actinomycetes. Only count plates with 30-200 colonies per plate.

To grow cultures of specific individual bacteria, select discrete colonies from any of the plates, choosing colonies that are well separated from neighboring colonies. Sterilize a spreading loop, then open the plate and touch the loop to an empty spot to cool. Next, pick a small amount of the colony of interest onto the loop.

Taking a fresh peptone-yeast plate, make a streak a few centimeters long on one side. Sterilize and cool again, then make a streak that crosses the initial streak only on the first pass. Repeat this process twice more in the same manner. This streaking “dilution” results in cells on the loop being separated from one another. Place the plate in a dark area to incubate at room temperature for two weeks.

From the bacterial and actinomycete colony counts, the Colony Forming Units per gram of soil can be determined. The number of colonies per gram of soil is equal to the number of colonies counted on the plate, multiplied by the reciprocal of the dilution plated. For example, if 46 colonies are counted on a dilution plate of 10-5 with a 0.1 mL inoculant, then the CFU per gram of soil is equal to 10-6 by 46, or 46 million CFUs per gram of soil.

Performing bacterial counts and cultures is a key first step in many scientific investigations or protocols.

Healthy soil contains between 106 and 108 bacteria per gram. Bacterial enumeration of soil can be used to determine the health of soils of interest, and counts of less than 106 and 108 bacteria per gram indicate unhealthy or poor soil. This may be caused by a lack of nutrients or organic matter, abiotic stress due to extreme soil pH, or soil contamination.

Many types of antibiotics used in medicine today were first identified from soil dwelling bacteria or fungi. Isolating pure strains from soil cultures can help in potentially help scientists identify new antibiotic compounds. Here, test bacteria or isolated compounds of interest can be added to plates grown with bacterial lawns, and their effects on the lawn growth recorded. Clear patches in bacterial lawns where growth was inhibited by the test bacteria or compound may indicate antibiotic activity.

Leguminous plants including peas and beans rely upon symbiotic relationships with nitrogen-fixing bacteria. These bacteria live freely in the soil or within nodules in the root system, and produce nitrogen compounds that are utilized by the plant. In poor soils, supplementing leguminous crops with cultured nitrogen-fixing bacteria from soils may boost the growth and health of the plants. This can result in bigger, hardier plants, which in turn give greater crop yield.

You’ve just watched JoVE’s introduction to bacterial enumeration. You should now understand how to perform dilutions of soil samples, how to plate for colony counting and colony isolation, and how to calculate the numbers of bacteria in soil samples. Thanks for watching!

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JoVE Science Education Database. JoVE Science Education. Culturing and Enumerating Bacteria from Soil Samples. JoVE, Cambridge, MA, (2023).