Introduction to Basic techniques for the detection of the  presence/absence of microorganisms

Introduction

Several tests used in the microbiological examination of foods are qualitative in nature ( presence/absence), including tests for the detection of Salmonella, Listeria monocytogenes,  Yersinia enterocolitica,  Campylobacter, Vibrio cholerae and  Vibrio parahaemolyticus. All these tests use the same basic microbiological techniques, which are  enrichment in one or more specific broths and subsequent isolation in solid culture media. The main reason why these tests are qualitative lies in the fact that the enrichment step makes quantification difficult, though possible, if indispensable, by using the MPN technique in these cases. For that purpose, it is necessary to repeat the same presence/absence test with several aliquots of the same sample, at least nine, in a multiple dilution test, or with five aliquots, in a single dilution test. Because of this, quantification requires a tremendous amount of work, in addition to being excessively expensive, two features that make quantification unnecessary and unjustifiable in most situations.

1.1  Enrichment

Enrichment is a critical step of presence/absence tests for three reasons. The first reason is that the population of pathogens in the samples is normally low (much below the detection limit of plate counts), making it necessary to increase the number of cells to detectable quantities. Products with a population of less than one bacterial cell per 100 g are not uncommon, and there are cases in which populations as small as one cell per 500 g of product have been detected. The second reason is that in most industrially processed foods, the cells of the target microorganism are injured by processing, thus requiring the recovery of the injured cells. Injured cells require a period of time under optimal growth conditions to reactivate the metabolic pathways responsible for multiplication. The third reason is that, normally, the competing microflora in the sample is present in much higher numbers than the target microorganism, making it necessary to inhibit the growth of this population in order to give the target an opportunity to multiply.

Enrichment may include one or more steps, depending on the target microorganism. In the Salmonella tests, for example, enrichment is done in two steps. When there are two steps it is common to call the first pre-enrichment or primary enrichment, while the second is called selective enrichment.

1.1.1 Pre-enrichment

The objective of pre-enrichment is to repair injured cells, offering conditions for their recovery, but at the same time, without favoring too much the growth of competing microflora. In general, injured cells do not grow under highly selective conditions, and for that reason, pre-enrichment broths are normally either not selective or only moderately selective. The pH of the medium must be in the optimum growth range of the target microorganism and, after the inoculation of acid products, should be adjusted to return to the optimal range, in case any alteration has occurred. For micro-organisms the optimal pH of which falls outside of the neutral range (Vibrio cholerae, for example, with an optimal pH value in the alkaline range), the pH of the broth may constitute a competitive advantage over the  accompanying microflora. The incubation temperature should also be in the optimal range, but incubation time should be just enough for restoring injuries. During the recovery phase, multiplication of the injured cells is minimal, and if incubation lasts longer than necessary, the competing microbial populations may increase excessively, thereby making later detection of the target more difficult or even impossible.

1.1.2  Selective enrichment

The objective of selective enrichment is to inhibit the competing microflora present in the samples, favoring at the same time multiplication of the target microorganism. This is achieved by using selective agents and/or restrictive conditions for the growth of the competing microflora, which may include: the pH of the culture medium, the temperature and/or atmosphere of incubation, the addition of antibiotics (polymixin B, ampicillin, moxalactam, novobiocin, D-cycloserine, oxytetracycline, vancomycin, trimethoprim, cyclohex-imide) and the addition of chemical compounds ) brilliant green, sodium selenite, bile salts, potassium tellurite, sodium lauryl sulphate). The nutritional composition of the medium should be optimal and, if possible, contain nutrients preferred by the target microorganism, such as uncommon carbon sources (D-manose, sorbitol, sodium citrate, for example). Not always the enrichment broths are able to provide an ideal balancing between the need to inhibit competitors without inhibiting the target microorganism. Eventually, some strains of the target may be sensitive to the selective conditions present, and in these cases, it is common to use more than one selective enrichment medium. This is the case, for example, of the  Salmonella test, which utilizes two broths.

1.2    Isolation in solid media (selective differential plating(

Once multiplication in enrichment broth(s) is achieved, it becomes necessary to differentiate and separate the target microorganism from the competing microflora. This is done by inoculating the culture on a solid medium, which also allows obtaining pure cultures to be later used in tests to confirm microbial identity. In general, isolation media are selective and differential to suppress part of the competing microflora and distinguish the target from the remaining microorganisms.

The selective agents used in solid media are the same as those used in liquid enrichment media, selected in function of the test. The most commonly used differential agents are the pH indicators, to differentiate the microorganisms that produce acids or bases during growth from those that do not. pH indicators change color in certain pH ranges, with phenol red and bromcresol purple being the most used pH indicators in culture media. Hydrogen sulphide (H₂S) indicators are also frequently used to differentiate microorganisms that produce this compound from those that do not in the metabolism of sulphur-containing amino-acids. _H2S indicators are iron-containing compounds, such as ferric citrate, ammonium ferric citrate or ammonium ferric sulphate. By reacting with H₂S, these compounds produce ferrous sulphide, a black and soluble precipitate that diffuses into the growth medium causing blackening of the medium. Other differentiating agents are egg yolk, to differentiate microorganisms that produce lypolitic enzymes from those that do not, and esculin, a naturally occurring glucoside used to differentiate the microorganisms that produces β-glucosidase. Hydrolysis of esculin yields D-glucose and esculetin (6,7-dihy-droxycoumarin), the latter compound being detected by the formation of a brown/black complex in the presence of iron salts.

Exactly like in the case of selective enrichment, some strains of the target microorganism may be sensitive to the selective conditions of plate culture media. In these cases, it is common practice to use more than one medium, such as in the case of the Salmonella test, for example, in which two or three are used.

1.3 Confirmation

This step aims at confirming the identity of an isolated culture, through tests that verify typical characteristics of the target microorganism(s). Most commonly used for microorganism confirmation are the morphological, biochemical and serological characteristics. The morphological characteristics include mainly the shape of the cells (cocci, straight rods, curved rods, spiral-shaped, etc), cell arrangement (isolated, in pairs, tetrads, in chains, in clusters, in filaments), in addition to Gram-stain, motility or spore-formation. The serological characteristics include principally verification of the presence of somatic O- antigens in the cell wall and flagellar H-antigens. The biochemical characteristics depend on the target microorganism and are presented in the corresponding specific chapters. The most commonly used tests are described below (Mac-Faddin, 1980):

1.3.1  Catalase test

The objective of the catalase test is to verify whether the bacterium is capable or not to produce the enzyme catalase, responsible for degrading hydrogen peroxide (H₂O₂). Hydrogen peroxide is a metabolite formed during the aerobic utilization of carbohydrates. It is toxic and can cause the death of the cells if it is not rap-idly degraded or decomposed. The degradation of this material occurs through the action of enzymes classified as hydroperoxidases, which include peroxidase and catalase (hydrogen peroxide reductase). The majority of the aerobic and facultative anaerobic bacteria that contain cytochrome also contain catalase. The majority of anaerobic bacteria, such as Clostridium sp, for example, contain peroxidase instead of catalase.

1.3.2  Citrate test

The objective of the citrate test is to verify whether the bacterium is able to use citrate as the sole source of car-bon for its growth. In the test, the only source of carbon available in the culture medium is citrate. If the bacterium has the metabolic apparatus necessary to assimilate citrate, multiplication will occur. Growth can be observed visually, by the formation and accumulation of a cell mass or by the color change of a pH indicator. If negative, there will be no growth.

1.3.3  Amino acid  decarboxylation tests

The objective of these tests is to verify whether the bacterium is able to decarboxylate amino acids forming amines, with the consequent alkalinization of the culture medium. Decarboxylation depends on the availability of decarboxylation enzymes, specific for each individual amino acid. Only the amino acids that have at least one chemically active group, in addition to the amine and carboxyl groups, can be subjected to or are capable of decarboxylation. The availability of one or more decarboxylases varies among the species and constitutes an interesting characteristic for differentiation purposes. The decarboxylases most commonly used in identification tests are arginine decarboxylase, lysine decarboxylase and ornithine descarboxilase, inducible enzymes produced by the bacteria only in the presence of the respective amino acids and under acidic conditions. The metabolism of descarboxylation is an anaerobic process that results in cadaverine and CO₂ produced from lysine and putrescine and CO₂ from ornithine. The metabolism of arginine is more complex and involves two metabolic pathways which may operate either simultaneously or separately: the arginine decarboxylase pathway and the arginine dehydrolase pathway. In the decarboxylase pathway, arginine is initially degraded to agmatine, which, at its turn, will be subsequently degraded to putrescine and urea by the agmatinase enzyme. In the urease-positive bacteria, the urea will then be further decomposed into two ammonia molecules. In the dehydrolase pathway, arginine is initially degraded to L-citrulline which, on its turn, will be further on degraded to L-ornithine, CO₂ and NH₃, by the enzyme citrulline ureidase. Irrespective of which pathway is used by the bacterium in the metabolism of arginine, the final products will be alkaline and will produce the same test result.

1.3.4  Phenylalanine deaminase test

The objective of this test is to verify whether the bac-terium is able to deaminate the amino acid phenylalanine. Deamination occurs in the presence of oxygen, by action of an amino acid oxidase, a flavoprotein that catalyzes the conversion of a molecule of phenylalanine into a molecule of phenylpyruvic acid and a molecule of ammonia. Phenylpyruvic acid may be detected in the culture medium by adding ferric chloride, which reacts with phenylpyruvic acid forming a colored compound, phenylhydrazone.

1.3.5  Carbohydrate fermentation tests

The carbohydrate fermentation tests aim at verifying whether a bacterium is able to ferment certain carbohydrates, producing acid with or without gas. Metabolic fermentation pathways of different carbohydrates are characteristic of species, being one of the most used phenotypic characters used for identification of bacteria. Fermentation processes are sequences of oxido-reduc-tion reactions that transform glucose into one or more carbon compounds, generating energy at the end of the reactions or process. The type(s) of final product(s) of glucose oxidation, that is, the type of fermentation, vary from species to species, and include acids, alcohols, CO₂ and H₂. The introduction of a carbohydrate different from glucose in the fermentation processes depends on the capacity of each species to insert the carbohydrate into the cell, convert it into glucose and then proceed with fermentation. The term “carbohydrates”, generally includes the compounds listed below, in addition to inositol, which, although it is a compound derived from non-carbohydrate sources, is also tested in the fermentation tests:

•  Monosaccharides: Ribose, Xylose, Arabinose (pentoses), Glucose, Fructose, Galactose (hexoses)

•  Disaccharides: Maltose (glucose + glucose), Sucrose (glucose + fructose), Lactose (glucose + galactose)

•  Trisaccharides: Raffinose

•  Polysaccharides: Starch, Inulin

•  Sugar-alcohols: Adonitol, Dulcitol, Mannitol, Sorbitol

1.3.6  Indole test

The objective of the indole test is to verify whether a bacterium is able to deaminate the amino acid tryptophan. Deamination of tryptophan depends on the availability in the bacteria of the tryptophanase enzyme system, which deaminates tryptophan producing indole, pyruvic acid, ammonia and energy. The indole released into the culture medium may be detected by a chemical reaction with the aldehyde present in the Indole Kovacs reagent used to perform the indol test (p-dimethylamino benzaldehyde), which will result in the formation of colored condensation products. These compounds, of a red-violet color, are concentrated in the alcohol phase of the reagent, forming a ring on the surface of the liquid culture medium.

1.3.7  Malonate test

The objective of the malonate test is to verify whether the bacteria is capable of using sodium malonate as car-bon source for its growth, resulting in alkalinization of the culture medium. Malonate is a compound that has the characteristic of inhibiting the activity of the succinate dehydrogenase enzyme, which is essential to the metabolic activity of the bacteria for the production of energy. Depending on the concentration of malonate present, multiplication of the microorganisms may be partially or completely inhibited, unless the bacterial cells are capable of using malonate itself as source of carbon and energy. When a bacteria is able to use malonate, it will also be able to use ammonium sulfate as sole source of nitrogen, generating sodium hydroxide as final product of metabolism. In this way, by adding ammonium sulfate to the culture medium, malonate-positive bacteria will cause an increase in the pH value of the medium, as a result of the build-up of sodium hydroxide. This alkalinization can be detected with bromotimol blue, a pH-indicator that changes color at pH 7.6.

1.3.8  Oxidation/Fermentation test ( O/F)

The objective of this test is to verify the type of metabolism of carbohydrate used by the bacterium or the non-use of carbohydrates. The use of carbohydrates for energy production in the bacteria may occur by two processes, the oxidative (cellular respiration) and the fermentative process. Oxidative metabolism is an aerobic process for most bacteria, that is, it only occurs in the presence of oxygen as final electron acceptor, although some bacterial species are capable of growing by replacing the oxygen by inorganic compounds such as nitrate and sulfate (anaerobic respiration). Fermentative metabolism, on the other hand, is an anaerobic process that does not depend on the availability of oxygen. The bacteria that use the oxygen-dependent oxidative metabolic process are called strictly aerobic bacteria or obligate aerobes, since their growth will only occur in the presence of O₂. The bacteria that use both the oxidative as the fermentative metabolic processes are called facultative anaerobes, because they can grow both in the presence and in the absence of O₂. The carbohydrate that is normally added to the growth media used to perform the oxidation/fermentation test is glucose, because it is used by most bacterial species. If the metabolism of glucose is strictly oxidative or oxidative and fermentative, that of the others will also be, making it unnecessary to test one by one. On the other hand, some bacteria are incapable of using glucose, although they do have the capacity to use other carbohydrates. There are also bacteria that are unable to use any kind of carbohydrate, such as Campylobacter, for example, which obtains its energy from amino acids or from intermediates of the tricarboxylic acid cycle.

1.3.9  Oxidase test

The objective of this test is to verify the presence of the cytochrome C enzyme, one of the oxidases that participate in the oxidative process of cellular respiration. The metabolism of cellular respiration of aerobic and facultative anaerobic bacteria presents, as the final step of the process, the electron transport system. This sys-tem is a sequence of oxidation-reduction reactions, in which electrons are transferred from one substrate to another, accompanied by the production of energy.

At the end of the chain, the final electron acceptor –which oxidizes itself to keep the system working – is oxygen, which receives the electrons transported from the substrates located at earlier points of the chain. For the electron transfer to oxygen to take place, the participation of a special group of electron transporting enzymes is necessary. These electron transporting enzymes, such as cytochrome oxidases, are found in all bacteria that use respiratory metabolism. The type and number of cytochrome oxidases present in the electron transportation chain of the different bacteria is a typical characteristic of each bacterial species and, hence, is used as a character for species identification. The oxidase test detects specifically one of these oxidases, the cytochrome C oxidase, which is not encountered in all bacteria capable of using respiratory metabolism. The reagent used in the test is always an artificial reducing agent that acts as final acceptor of the electrons transferred by the cytochrome oxidase C enzyme. These reagents have the characteristic to change color when passing from the reduced state to the oxidized state, in a way such that, when they receive the electrons, they oxidize themselves, bringing about a clearly visible color reaction.

1.3.10  Nitrate reduction test

The objective of this test is to verify whether a bacterium is able to reduce nitrate to nitrite or free nitro-gen gas. The reduction of nitrate (NO₃−) is usually an anaerobic process and the final product of the reduction reaction varies as a function of the bacterial species, and may include nitrite (NO₂−), ammonia (NH₃), nitrogen gas (N₂), nitric oxide (NO), nitrous oxide (N₂O) and hydroxylamine (R-NH-OH). The most common are nitrite and, in the case of bacteria that are also capable of reducing nitrite, free nitrogen gas that results from this reduction reaction. Complete reduction from nitrate to nitrogen gas (via nitrite or nitrous oxide) is called denitrification. The reduction products may be used or not in the metabolism of the bacteria, depending on the environmental conditions. When not used, they are excreted to the culture medium, where they may be detected. Nitrite may be detected through a color reaction with the reagents used for nitrate tests, a mixture of  α-naphthylamine and sulphanilic acid, which react with the nitrite form-ing a colored compound. Alternatively, if the medium is free of nitrate reduction products, the occurrence of the process can be proven by the absence of the nitrate originally added to the culture medium. This verification is accomplished with zinc, which is capable of causing a color reaction with any nitrate present in the medium, indicative of non-reduction. If the reaction does not occur, it may be concluded that the nitrate was reduced.

1.3.11  Urease test

The objective of the urease test is to verify whether the bacterium produces the urease enzyme, responsible for decomposing urea into ammonia. Urease is an enzyme of the amidase type, which catalyzes the hydrolysis of amides such as urea. The hydrolysis of each urea molecule results in two ammonia molecules, which increase the pH of the culture medium and may be detected by phenol red, a pH indicator that changes color at pH 8.4.

1.3.12  Methyl Red test ( MR)

The objective of the methyl red test is to verify whether the fermentative metabolism of the bacterium is of the mixed-acid type. Fermentation of glucose by the bacteria may result in different final fermentation products, with the type of fermentative metabolism being a characteristic of the species. In mixed-acid fermentation, the final product is a mixture of acids (2 glucose + 1 H₂O ⇒ 2 lactic acid + 1 acetic acid + 2 formic acid + 1 ethanol) which reduce the pH of the medium to a value lower than 4.5. This pronounced reduction in pH, and which exceeds the buffering capacity of the phosphate buffer present, may be detected by adding to the culture a few drops of a methyl red solution, a pH indicator that changes color below 4.5.

1.3.13  Voges-Proskauer test ( VP)

The objective of this test is to verify whether the bac-terium produces butylene-glycol (butanediol) as final fermentation product of glucose. In butylene glycol fermentation, the final product is preceded by an intermediate precursor, acetoin (acetyl methyl carbinol), converted into butilene-glycol by action of diacetyl reductase. Acetoin may be detected in the VP test, by the addition of the reagents for VP test.

References

Silva, N.D .; Taniwaki, M.H. ; Junqueira, V.C.A.;  Silveira, N.F.A. , Nasdcimento , M.D.D. and Gomes ,R.A.R .(2013) . Microbiological examination methods of food and water a laboratory Manual. Institute of Food Technology – ITAL, Campinas, SP, Brazil .

MacFaddin, J.F. (1980) Biochemical tests for identification of medical bacteria. 2nd edition. Baltimore, Williams & Wilkins. Sperber, W.A., Moorman, M.A. & Freier, T.A. (2001) Cultural methods for the enrichment and isolation of microrganisms. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 5, pp. 45–51.