低濃度細(xì)菌計數(shù)常采用多管技術(shù)，而普通的活菌計數(shù)則通常采用菌落計數(shù)法（見5.1)。一般情況下，檢測食品中的細(xì)菌總數(shù)時，因為不了解食品中細(xì)菌的種類，所以很難選擇適當(dāng)?shù)呐囵B(yǎng)基和培養(yǎng)條件。因不同細(xì)菌的營養(yǎng)和生理條件不同，也就不可能獲得真實的活細(xì)菌數(shù)值。
一個樣品中的細(xì)菌種類可能有專性需氧菌、專性厭氧菌、兼性需氧菌等，其中，有些細(xì)菌在37℃生長良好，但不能在20℃生長；有些細(xì)菌在20℃生長良好，但不能在37℃生長，而另一些菌在這兩種溫度條件下或許均能生長。即使用四套平皿同時培養(yǎng)——第一套在20℃培養(yǎng)需氧菌,第二套在37℃培養(yǎng)需氧菌，第三套在20℃培養(yǎng)厭氧菌，第四套在37℃培養(yǎng)厭氧菌，也不可能得到活菌總數(shù)，因為在培養(yǎng)時會有一些未知菌生長且被重復(fù)計數(shù)。所以,不應(yīng)將需氧嗜溫菌計數(shù)作為細(xì)菌的總數(shù)——這并不符合事實。
在選擇培養(yǎng)基時，也會出現(xiàn)類似的問題。菌落計數(shù)法和多管計數(shù)法可用于檢測適當(dāng)
培養(yǎng)條件下有活性的細(xì)菌數(shù)。也就是說,菌落的生長(瓊脂培養(yǎng)基內(nèi)或表面)或試管的渾濁僅能說明細(xì)菌在該培養(yǎng)條件下具有活性，而不代表其在加工過程或消費環(huán)境下具有活性。這個差別非常重要，例如當(dāng)細(xì)菌在加工或貯存過程中被破壞或損傷時，將食品樣品用細(xì)菌總數(shù)(顯微鏡鏡檢）和活菌計數(shù)兩種方法同時檢測，若細(xì)菌總數(shù)高而活菌數(shù)低,并不一定就意味著顯微鏡觀察到的多數(shù)細(xì)菌已經(jīng)死亡，也可能只是因為這些細(xì)菌在計數(shù)培養(yǎng)基上不能增殖。
食品微生物工作者必須根據(jù)工作經(jīng)驗選擇一種或多種培養(yǎng)條件，一般而言，應(yīng)該選擇大多數(shù)樣品都能得到最高計數(shù)的培養(yǎng)條件。在大多數(shù)情況下可供選擇的培養(yǎng)條件如下：
0-10℃ 嗜冷菌汁數(shù)
20-30℃ 嗜中溫菌計數(shù)
35——37℃（或45℃） 恒溫動物的共生菌或寄生菌的計數(shù)
55-63℃或更高溫度 嗜熱菌計數(shù)
嗜冷菌的計數(shù)最好采用表面計數(shù)技術(shù)，這樣可避免將細(xì)菌置于熱瓊脂當(dāng)中。真正的
嗜冷菌除寄生在海產(chǎn)品當(dāng)中外，不能在其他食品中存活，而且一旦暴露在周圍的環(huán)境中就會死亡。如果懷疑有嗜冷菌存在，樣品稀釋液和平皿必須先存放在10℃以下預(yù)冷。
培養(yǎng)基成分越復(fù)雜，能夠生長的細(xì)菌種類越多。因此，在葡萄糖胰蛋白胨酵母浸膏瓊脂上生長的細(xì)菌種類比營養(yǎng)瓊脂多的多。用營養(yǎng)球脂進(jìn)行凍生肉的活菌計數(shù)，結(jié)果小于10000個/g。而用平板計數(shù)瓊脂計數(shù)，結(jié)果為每克幾百萬個。之所以如此,是因為在食品中占優(yōu)勢的菌群是腸道球菌，它在有葡萄糖存在的條件下才能生長。對于某些樣品，在培養(yǎng)基中增加血清,蛋黃或牛奶等可提高計數(shù)結(jié)果。但通常不添加這些物質(zhì)，因為這樣要增加費用，而且使培養(yǎng)基的制備過程更為復(fù)雜(血清、蛋黃必須在倒平板前迅速加入）。
如果要分離腐生菌，那么培養(yǎng)基至少應(yīng)部分模擬寄生菌的棲息環(huán)境。例如添加0.1%
脫脂乳的平板計數(shù)瓊脂常用于日常用品的檢測。此外，培養(yǎng)溫度應(yīng)與食品的儲存溫度類似。為了找到合適的培養(yǎng)條件，可用顯微鏡直接對稀釋液進(jìn)行檢測。如果在顯微鏡下觀察到某特定形態(tài)的細(xì)菌，而在分離培養(yǎng)基上未分離到該菌，則很可能是因為分離環(huán)境不適合該類細(xì)菌的生長。很多情況下，食品或其他環(huán)境下的典型細(xì)菌不能夠生長都是由于沒有在培養(yǎng)基中添加食品成分或適于細(xì)菌在該環(huán)境下生存的成分，Varnam和Grainger給出了一個比較顯微鏡檢測和平板分離細(xì)菌的好例子。他們在用顯微鏡檢測咸肉腌制水中的優(yōu)勢菌時，發(fā)現(xiàn)革蘭氏陰性桿菌占優(yōu)勢。但是，在用培養(yǎng)基檢測時,必須在培養(yǎng)基中加人18%——20%的氯化鈉和15%?20%過濾除菌的豬肉浸汁才能檢測到這些革蘭氏陰性桿菌。他們隨后得出結(jié)論，豬肉浸汁并不是培養(yǎng)基中必須的營養(yǎng)成分，其作用是除掉培養(yǎng)基中的過氧化物。
因此，建議可用二氧化錳代替豬肉浸汁。
對于用蒸餾水（而不是海水）配制的培養(yǎng)基，許多海洋細(xì)菌則不能生長。
在研究某棲息地中的微生物組成時，需經(jīng)常對添加成分的有效性進(jìn)行測試。但解釋
或比較某些細(xì)菌的形態(tài)學(xué)特征時必須小心，因為它們（如棒桿菌）在樣品和培養(yǎng)基中呈現(xiàn)出的形態(tài)學(xué)特征對能不同。
對含有霉菌和酵母菌的樣品進(jìn)行細(xì)菌計數(shù)的培養(yǎng)基中，必須添加抗真菌的抗生素，如可添加10mg/kg的環(huán)己酰亞胺。
Bacterial count is often used for low concentration bacteria counting, while colony counting is usually used for counting common live bacteria (see 5.1). In general, when detecting the total number of bacteria in food, it is difficult to choose suitable media and culture conditions because they do not know the kinds of bacteria in food. Because of the different nutritional and physiological conditions of different bacteria, it is impossible to obtain real viable bacterial counts.
There may be specific aerobic bacteria, specific anaerobes and facultative aerobic bacteria in one sample. Among them, some bacteria grow well at 37 degrees, but can not grow at 20. Some bacteria grow well at 20, but can not grow at 37, while others can grow at these two temperatures. At the same time, four sets of flat plates were used at the same time - the first set of aerobic bacteria was cultured at 20 centigrade, second sets of aerobic bacteria were cultured at 37 C, third sets of anaerobic bacteria were cultured at 20 centigrade, fourth sets of anaerobic bacteria were cultured at 37 degrees C, and the total number of living bacteria could not be obtained, because some unknown bacteria were long and counted repeatedly during the culture. Therefore, it is not appropriate to count aerobic aerobic bacteria as the total number of bacteria.
Similar problems arise when selecting medium. Colony counting method and multi tube counting method can be used for detection.
The number of active bacteria under culture conditions. That is to say, the growth of the colony (in the agar culture or the surface) or the turbidity of the tube only shows that the bacteria are active under this culture, but do not represent their activity in the process of processing or in the consumption environment. This difference is very important, for example, when bacteria are damaged or damaged during processing or storage, the total number of bacteria in the food sample (microscopic examination) and the living bacteria count are detected at the same time. If the total number of bacteria is high and the number of living bacteria is low, it does not necessarily mean that most of the bacteria observed by the microscope are dead, and it may also be possible. Only because these bacteria can not proliferate on counting medium.
Food microorganism workers must choose one or more culture conditions according to their work experience. Generally speaking, most of the samples should be selected to get the highest count of culture conditions. In most cases, the following conditions are available:
The number of cold eosinophils at 0-10
Count of thermophilic bacteria at 20-30 degrees centigrade
Enumeration of symbiotic bacteria or parasitic bacteria in 35 - 37 C (or 45 C) animals at constant temperature
The count of thermophilic bacteria at 55-63 or higher temperatures
It is better to use the surface counting technology to prevent the bacteria from being placed in the thermal agar. Real
In addition to being parasitic in marine products, cold resistant bacteria can not survive in other foods, and they will die once exposed to the surrounding environment. If suspected chilling bacteria exist, the sample diluent and pan must be stored below 10 C for pre cooling.
The more complex the medium is, the more bacteria can grow. Therefore, there are more species of bacteria growing on glucose tryptone yeast extract agar than nutrient agar. The count of viable bacteria for frozen meat using nutrient ball fat was less than 10000 /g. The count of agar plates was used to count millions of grams per gram. The reason is that the dominant bacteria in food are Enterococcus, which can grow under the condition of glucose. For some samples, the increase of serum, egg yolk or milk can increase the counting results. However, these substances are not usually added because it increases the cost and makes the preparation of the medium more complex (the serum and yolk must be quickly added before the flat plate).
If the saprophytic bacteria are to be separated, the culture medium should at least partially simulate the habitat of the parasite. For example, add 0.1%
Plate count agar for skim milk is commonly used for routine products. In addition, the incubation temperature should be similar to the storage temperature of food. In order to find suitable culture conditions, the diluent can be directly detected by microscope. If a particular form of bacteria is observed under a microscope and the bacteria are not isolated on a separate medium, it is likely that the separation environment is not suitable for the growth of this kind of bacteria. In many cases, typical bacteria in food or other environments are not able to grow because they do not add food ingredients in the medium or are suitable for bacteria to survive in the environment. Varnam and Grainger give a better example for comparing microscopes and separating bacteria from the plate. They found that gram-negative bacilli were dominant when they examined the dominant bacteria in salted meat by microscopic examination. However, it is necessary to detect these gram-negative bacilli by adding 18% to 20% sodium chloride and 15% to 20% filtrated pork dipping in the culture medium. They concluded that pork juice was not the necessary nutrient in the culture medium, and its function was to remove peroxide from the culture medium.
Therefore, it is suggested that manganese dioxide be used instead of pork juice.
Many marine bacteria can not grow on distilled water (not seawater).
When studying the composition of microorganisms in a habitat, it is necessary to test the effectiveness of added ingredients frequently. But explain
It is important to be careful when or comparing the morphological characteristics of certain bacteria, because they are different in the morphology of the samples and in the medium.
In the culture medium containing bacteria and yeast, bacteria must be added with antifungal antibiotics, such as cycloheximide, which can be added to 10mg/kg.
