Scientific experiments are inseparable from instruments. Although human eyes have 576 million pixels, we still can't see objects that are thin enough or moving fast enough. In the past, biology was roughly divided into zoology and botany. Since the appearance of the microscope in the 1760s, the discipline of microbes has been established. In fact, every reform and progress in history is related to the invention of new instruments. The leaders of our school attached great importance to the experiment. In 2005, a new laboratory building was built, and a large number of advanced instruments were purchased to support the experimental teaching work of the teachers, which enabled the school to successfully build a biological clean room, a molecular biology laboratory and a microscopic digital interactive laboratory.

The purpose of the Microbiology Lab is to train students to master the most basic operational skills of microbiology; to understand the basics of microbiology; to deepen understanding of some of the microbiology theories taught in the classroom. At the same time, through experiments, students can understand and use experimental equipment and equipment, cultivate students' ability to observe, think, analyze and solve problems, cultivate students' realistic and serious scientific attitude, and work hard to save and protect public goods.

First, our school's microbiological experimental hardware equipment

Our microbiology laboratory equipment is basically complete, with biological clean room, molecular biology laboratory and microscopic digital interactive laboratory, which can meet the needs of high school microbiology experiment operation. The Bio Clean Room was built in 2009 and features a buffer room, a culture room, a transfer window and an operator room. The operating room has an ESCO biosafety cabinet and five ultra clean benches. Aseptic operation is inseparable from molecular experiments, so the biological clean room is adjacent to the molecular biology laboratory, which is convenient for experimental operation and scientific management. Among them, large and medium-sized instruments include intelligent artificial climate chamber, carbon dioxide incubator, vertical pressure steam sterilizer, electric heating constant temperature blast drying box, water-isolated constant temperature incubator, constant temperature shaking incubator, and the like. Small instruments include pipettes, electronic balances, shakers, water baths, electric stoves, induction cookers, microwave ovens, and more. Of course, a variety of consumables are also included, such as a pipette tip, an inoculating loop, an applicator, a conical flask, a test tube, a petri dish, a culture medium, and the like.

Second, the gradual experimental course design

The microbial experiments related to high school biology compulsory 3 "steady state and environment" and elective 1 "biotechnology practice" were improved. Based on the principle of shallowness and deepness, a series of microbial experiments were designed to students step by step. The preparation of microbiology experiments is complicated and takes a long time. Therefore, for the high school students microbiology experiment class, we set up a biological experiment group to use the school's school-based curriculum every Wednesday (two hours per class) for experimental exploration.

Teachers use the courseware and field visits through the laboratory to give students a preliminary understanding of the commonly used instruments and aseptic techniques for microbiological experiments. Glassware, culture medium, etc. used in microbiology experiments are to be sterilized. Microbiology experiments must master aseptic technique, and “writing labels” during the experiment is also a very important step. In this part of the knowledge class, the teacher has already taught, so we use a class to revisit the common sense of microbiology experiments, and then lead the students into the microbiology laboratory to visit and understand, and use the perceptual knowledge to deepen the understanding of microbiology knowledge.

1. Examination of laboratory environment and human surface microbes

Microbiology experiments are a rigorous study. Because microorganisms are invisible to the naked eye, students often do not know that the environment and all parts of the body that come into contact with the outside world have microbes. In fact, they are widely distributed in indoor and outdoor air, water and soil, on the surface of tables and chairs, clothing and skin, mucous membranes (such as mucous membranes of nasal cavity, mouth, throat, etc.). Therefore, microorganisms can be said to be The seams are not drilled and are not perforated. For middle school students, our experiment aims to establish the concept of “microbes everywhere” and emphasizes that a key difference between microbiology experiments and other experiments is the concept of “aseptic operation”. Therefore, we put the experiment "inspection of laboratory environment and human surface microbes" in the first place.

The experimental procedure was such that the microorganisms in different environments were inoculated with nutrient agar medium using the following method:

(1) Air, open the sterile plate and expose it to normal laboratory air and clean room air for 30 to 60 minutes.

(2) The table top, with a sterile cotton swab, first wet in an area of ​​the sterile plate, then wipe the table with it, and then use this cotton swab to scribe back and forth in another area of ​​the plate.

(3) Hair, on top of the sterile plate that uncovers the lid, shake the hair vigorously several times by hand to allow the bacteria to land on the surface of the plate, and then cover the lid. (4) Fingers, with unwashed hands on the surface of one side of the sterile plate, gently back and forth; after washing hands with detergent, gently back and forth on the other side of the surface. (5) Oral, open the lid of the sterile plate, align the surface of the plate medium with the mouth, cough or sneeze, and then cover the lid. Next, all the labeled detection plates were placed in an incubator and cultured at 37 ° C for 1 to 2 days, and then the number of microbial growth and colony morphology from different sources were observed and compared.

In the operation steps, the “write label” step needs to be repeatedly emphasized with the students. For any experiment, it is necessary to mark the vessel with a marker before the hands-on operation. The symbol of the dish is usually written on the bottom of the dish. If you write on the lid and observe the results of two or more Petri dishes at the same time, it is easy to confuse when you open the lid. Write the class, name, date with a marker. In this experiment, you should also write the source of the sample (such as laboratory air or sterile room air or hair, etc.), the word should be as small as possible, written on the side of the dish, not written in In the middle, so as not to affect the observation.

The equipment used in this experiment was mainly a vertical pressure steam sterilizer and a smart artificial climate chamber.

In this experiment, students realized the necessity of aseptic technique and environmental sanitation, and then knew to prevent the contamination of culture by external microorganisms. This is a must for the entire microbiological experiment course and for future microbiological experiments.

2. Laboratory culture and plate colony counting method for bacteria in water

Laboratory training of microorganisms, the purpose of which is to master the method of pouring the plate and the basic operation techniques of several isolated and purified microorganisms. At the same time, the laboratory culture of bacteria in water, learned the method of taking water samples and the method of plate colony technology.

Inverted plate technology: After sterilizing hands, under the ultra-clean workbench or around the alcohol lamp, hold the flask in the right agar medium, hold the plate in the left hand, open the bottle stopper and the bottle mouth and bottle next to the alcohol lamp. Plug, the bottle stopper is burned and placed next to the alcohol lamp, then the left hand will cover the culture dish with a slit near the flame, and quickly pour the medium into about 15mL. After capping, gently shake the culture dish to evenly distribute the culture dish. In view of the first operation of the student, it is more difficult to open the culture dish with one hand. We recommend that the student place the culture dish on the table near the flame, open the culture dish, inject the medium, shake it and then cool it to make the plate. .

For safety reasons, we use water from tap water and school water purifiers as experimental subjects. The experimental process is as follows:

(1) Take the water sample, open the faucet and let the water flow for 5 minutes. Then take the water sample in the sterilized triangle flask and label it.

(2) Plate coating method, using a pipette to draw 1 mL of water sample, inject it into a sterilized culture dish, and gently spread it on the surface of the medium with a sterile glass coating rod, and make one sample into three parallels.

(3) Culture, inoculate all the plates, and incubate them in an incubator at 28 ° C for cultivation.

(4) Bacterial plate counting method, after 7 days of plate culture, the number of colonies of the plate was observed and calculated, and the average number of colonies of the three plates was the total number of bacteria of 1 mL of water sample.

(5) Bacterial plate streak separation method, under the ultra-clean workbench, near the flame, take the bottom of the dish in the left hand, take the inoculation ring in the right hand, pick the above water sample and culture a single colony on the plate, then carry out the culture observation. Line results. There are many methods for scribing, but no matter which method is used for scribing, the purpose is to dilute the sample on the plate by scribing to form a single colony.

Separation of inverted plates and plates is the most basic operation technique for microbial experiments. Separation and purification of colonies often requires more than 3 times to separate single colonies. Therefore, doing microbial experimental research often requires a large number of plates and hundreds of thousands of separations and purifications. The large amount of work is large, and the preparation work takes a long time. In order to save the flattening time and the scribing time, we have created a multi-layer sterilization dish inverted plate technology and toothpick separation and purification technology, saving a lot of experiments. Time also prevents the clean room from being deprived of oxygen due to long-term burning of alcohol lamps.

The experiment was carried out in a clean room with a clean bench to allow students to master the use of pipettes, glass coating sticks, and inoculating loops.

3. Morphological observation of yeast and blood cell counting plate counting method

A few hours before the experiment, 5% sterile glucose water was added, followed by 0.5 g of edible yeast powder, and the yeast suspension was prepared, which was then observed and counted by microscopy. Cover the clean and dry blood cell counting plate with a cover slip, and then use a sterile fine-mouthed dropper to drip the bacterial suspension from the edge of the coverslip (not too much), so that the bacterial suspension penetrates the capillary along the gap. The function enters the counting chamber by itself, and the general counting chamber can be filled with the bacterial liquid. Be careful not to have bubbles. The morphology of the yeast microscope was observed using 5 minutes and then microscopic counting was started. If the bacterial liquid is too concentrated or too thin before counting, it is necessary to readjust the dilution and then count. Generally, the dilution of the sample requires 5 to 10 cells per small compartment. The cell count in 5 cells (optional 4 corners and the center cell) is selected for each counting chamber. The cells on the grid line are generally only on the upper and right lines. In the case of yeast germination, when the size of the bud reaches half of the mother cell, it is counted as two cells. The sample is counted from the values ​​counted in the two counting chambers to calculate the bacterial content of the sample.

This experiment was carried out in a micro-digital interactive laboratory equipped with 30 digital light microscopes for quick and easy observation, and students can also count bacteria on a computer screen.

4. Select the subject to explore

After the above series of experiments, we will divide the students into several interest groups, and carry out microbiology experiments in the laboratory with reference to the microbiological related experiments of 1 "biotechnology practice" or self-designed microbiology experiments. For example, Elective 1 Experiment 5 "Separation and Counting of Bacteria Decomposing Urea in Soil", Experiment 6 "Separation of Microorganisms Decomposing Cellulose", and Experiments for Observing and Cultivating Lactic Acid Bacteria in Yogurt.

Third, the conclusion

Using our functional laboratories and equipment, we have developed a series of basic microbiological experimental techniques for high school students. According to the idea of ​​“establishing the concept of aseptic-isolation and purification technology-colony counting method and microscopic counting method-self-designed subject”, students enter the biological clean room, molecular biology laboratory and micro-digital interactive laboratory, using ultra-clean Workbench, constant temperature incubator, shock incubator, sterilizer, digital microscope and other instruments. The relationship between experiment and instrument has a deeper understanding, popularizing the method of scientific experiment, and improving the spirit of scientific exploration of students.

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