КОМПЛЕКСНИЙ МЕТОД ВИЗНАЧЕННЯ СКЛАДУ МІКРОФЛОРИ КИШКОВИКА ЛАБОРАТОРНИХ ТВАРИН
The method of determination of qualitative and quantitative composition of intestinal microflora of laboratory animals, namely albino rats, was described. The method allows to simultaneously identify different groups of microorganisms in one sample of intestinal composition and to get a full picture...
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| Datum: | 2015 |
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| Format: | Artikel |
| Sprache: | Englisch Ukrainisch |
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Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine
2015
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| Online Zugang: | https://smic.in.ua/index.php/journal/article/view/137 |
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| Назва журналу: | Agriciltural microbiology |
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Agriciltural microbiology| _version_ | 1871465424361619456 |
|---|---|
| author | Камінська, М. В. |
| author_facet | Камінська, М. В. |
| author_institution_txt_mv | [
{
"author": "М. В. Камінська",
"institution": "Інститут біології тварин НААН"
}
] |
| author_sort | Камінська, М. В. |
| baseUrl_str | https://smic.in.ua/index.php/journal/oai |
| collection | OJS |
| datestamp_date | 2026-07-22T10:10:42Z |
| description | The method of determination of qualitative and quantitative composition of intestinal microflora of laboratory animals, namely albino rats, was described. The method allows to simultaneously identify different groups of microorganisms in one sample of intestinal composition and to get a full picture of microbocoenosis within 4 days that allows to consider animal’s health, dysbiotic infringement in intestinal microflora composition and its correction with probiotic preparations. |
| doi_str_mv | 10.35868/1997-3004.22.60-65 |
| first_indexed | 2025-07-17T12:24:02Z |
| format | Article |
| fulltext |
THE COMPLEX METHOD FOR DETERMINATION OF INTESTINAL MICROFLORA
COMPOSITION OF LABORATORY ANIMALS
M. V. Kaminska
Determination of qualitative and
quantitative composition of intestinal microflora
of the laboratory animals, namely albino rats is
important for conduct of the study regarding
assessment of the impact of probiotic
preparations for the restoration of intestinal
microbiocenosis of animals under the
experimental dysbiosis. There is no single
common method to establish this composition.
Normative documents establish methods for the
quantitative determination of the content of
specific groups of microorganisms [1-4], such as
E. coli, staphylococci, proteus, pathogenic
enterobacteria. However, the research work
should simultaneously determine different
groups of microorganisms in one sample of the
intestinal content to establish the overall picture
of infringements or correction of the composition
of intestinal microbiocenosis of the laboratory
animals.
The aim of the work was to develop and
provide generalizing method for establishment of
qualitative and quantitative composition of
microflora of the intestinal content in laboratory
albino rats.
Preparation of glassware and selective
media. Each sample requires preparation of
sterile weighing bottle (for entering sample of the
intestinal content of 1 g), sterile glass rod, 7
sterile spatula, 3 sterile test tubes, 1 mL (6 pcs.)
and 10 mL (for dilutions) sterile pipettes, 8 sterile
Petri dishes.
All glassware is sterilized in a steam
sterilizer for 30 minutes at a pressure of 0.75 atm.
and temperature of 120°C.
Selective media are prepared and
sterilized according to the instructions of the
manufacturer and poured into sterile Petri dishes.
Therefore, the following is prepared: 3 dishes
with Endo medium, 1 dish with bismuth-sulphite
medium, 1 dish with Sabouraud medium, 1 dish
with blood agar, 1 dish with Baird-Parker
medium. Blaurock medium is prepared as
semi-liquid in accordance with recipe [5], poured
into 2 test tubes of 4.5 mL and autoclaved with
cotton stoppers. Sterile slope agarized
Olkenytskyi and Simons media (10 of each for
each test tube) are prepared to identify individual
species of enterobacteria. Test tubes with slope
medium can be stored in the refrigerator for 30
days.
For microscopic examination of the
samples from Blaurock medium and suspicious
colonies from other media, set of reagents for
Gram staining and microscope are used.
Selection and preparation of samples.
Samples of rat intestinal content (commonly
from the rectum) are taken immediately after
slaughter. For this purpose, a region of intestine,
the content of which will be investigated is cut
with sterile scissors, and placed into a sterile Petri
dish. Further, applying aseptic regulations,
intestine is cut and its content is removed. 1 g of
content is placed in the sterile weighing bottle
and 9 mL of sterile normal saline solution is
added. Thus, ten-fold dilution (10-1) is obtained.
It is not infrequent that the intestinal
region has less content than one gram, especially
when working with sick animals. Then maximum
possible number of content is weighed, and
volume of normal saline solution to be included
in the weighing bottle is-recalculated for 10-fold
dilution. For example, when the sample weight is
500 mg, we are adding 4.5 mL of solution, and in
case of 300 mg – 2.7 mL of solution.
Weighing bottles with samples are not keep, and
immediately used for inoculation of media.
Before preparing dilutions, content of the
weighing bottle is thoroughly mixed with sterile
glass rod.
Preparation and inoculation of
dilutions.
To prepare the required dilutions, three sterile
test tubes are used in which 9.9 mL of sterile
normal saline solution is added.
Sample 0.1 mL of solution from the
weighing bottle with sterile pipette and transfer to
a test tube No. 1 with 9.9 mL of normal saline
solution, and then shake. Thus, we obtain 10-3
dilution. Transferring 0.1 mL of the solution from
this test tube to the next test tube (tube No. 2), we
obtain 10-5 dilution, and so on under the scheme
(Fig. 1).
After preparation of all dilutions,
withdraw 0.1 mL of the solution from each test
tube using sterile pipette and place into the Petri
dish with a corresponding selective medium,
increasing each dilution for another 10 times. The
solution is rubbed over the surface with a sterile
spatula until its complete absorption with agar.
Place 0.5 mL of the solution with certain
dilutions into the test tubes with Blaurock
medium, and at the same time dilution degree is
increased ten times.
Thus, according to the scheme (see. Fig. 1),
withdraw 0.1 mL of solution from the weighing
bottle and place into the Petri dish with Endo
medium and bismuth-sulphite agar, getting a 10-2
dilution. From the test tube No. 1 place 0.1 mL in
the dishes with Endo medium, blood agar,
Baird-Parker and Sabouraud agar. This resulted
in a 10-4 dilution. From the test tube No. 2 place
0.1 of solution into the dish with Endo medium
and 0.5 mL of solution into the test tube with
Blaurock medium obtaining 10-6 dilution.
Withdraw 0.5 mL of the solution from test tube
No. 3 into the test tube with Blaurock medium
and obtain 10-8 dilution.
Leave inoculated dishes and test tubes on
the table for 15 minutes, and then put in a
thermostat at 37°C. Dishes with Endo medium,
blood agar and bismuth-sulphite agar are
controlled 24 hours after inoculation, dishes with
Baird-Parker and Sabouraud medium – after 48
hours, and Blaurock medium undergoes
microscopy at day 4 of growth.
Evaluation of results. Determination of
qualitative and quantitative composition of
intestinal microflora of laboratory animals makes
it possible to judge the health of animal abuse
dysbiotic infringements in the composition of
intestinal microflora and their correction with
probiotics.
Thus, according to our studies [7-11],
normal value of the total number of E. coli in
laboratory rats is within 106-108 CFU/g, while the
number of weak-fermenting strains does not
exceed 25%. There are isolated lactose negative
strains. Among haemolytic microorganisms,
representatives of staphylococci, streptococci,
and E. coli are predominant. Number of
staphylococci is within 104-106 CFU/g, and the
number of pathogenic strains does not exceed
10% of the total number of coccal forms. The
most numerous group of microorganisms is
bifidobacteria and lactobacilli, which generally
account for about 99% of the total number of
microorganisms in the intestine of rats. Their
number is 108-1010 CFU/g for bifidobacteria and
106-108 CFU/g for lactobacilli. Reduction of their
number by two decades (100 times), at the
background of the growing number of
opportunistic groups of bacteria indicates the
occurrence of a serious intestinal dysbiosis.
Number of fungi of the genus Candida ranges
within 104-105 CFU/g. Increase in their number
by decade or more suggests occurrence of fungal
dysbiosis. Development of Proteaceae dysbiosis
is evidenced by the rise in the number of proteus
cells from 102-103 CFU/g to 104-105 CFU/g.
Total number of other lactose negative
opportunistic enterobacteria ranges from 102-103
CFU/g.
It should also be noted that study of
intestinal microflora of laboratory rats, which are
fed with probiotic supplements allows to predict
increase in the total number of bifidobacteria and
lactobacilli, therefore, it is required to make
additional dilution (test tube No. 4), and inoculate
Sabouraud medium with 10-10 dilution. In case of
dysbiosis modelling by administration of
antibiotics, we predict decrease in the total
number of E. coli or staphylococci (depending on
the type of antibiotic), and bifidobacteria and
lactobacilli. Therefore, inoculation on selective
medium for these types of microorganisms
should be done at lower dilutions.
Thus, we have proposed the complex
method for determination of intestinal
microbiocenosis of laboratory animals, which
generally takes 4 days, but makes it possible to
determine the qualitative and quantitative
composition of microflora content of rat rectum
and diagnose its infringements.
|
| id | oai:ojs2.smic.in.ua:article-137 |
| institution | Agriciltural microbiology |
| keywords_txt_mv | keywords |
| language | English Ukrainian |
| last_indexed | 2026-07-23T01:01:49Z |
| publishDate | 2015 |
| publisher | Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine |
| record_format | ojs |
| resource_txt_mv | smicinua/b9/2834fa0c19723946715b764e6497d6b9.pdf |
| spelling | oai:ojs2.smic.in.ua:article-1372026-07-22T10:10:42Z THE COMPLEX METHOD FOR DETERMINATION OF INTESTINAL MICROFLORA COMPOSITION OF LABORATORY ANIMALS КОМПЛЕКСНИЙ МЕТОД ВИЗНАЧЕННЯ СКЛАДУ МІКРОФЛОРИ КИШКОВИКА ЛАБОРАТОРНИХ ТВАРИН Камінська, М. В. method, intestinal microflora, rats метод, мікрофлора кишковика, щурі The method of determination of qualitative and quantitative composition of intestinal microflora of laboratory animals, namely albino rats, was described. The method allows to simultaneously identify different groups of microorganisms in one sample of intestinal composition and to get a full picture of microbocoenosis within 4 days that allows to consider animal’s health, dysbiotic infringement in intestinal microflora composition and its correction with probiotic preparations. Описано методику визначення якісного та кількісного складу мікрофлори вмісту кишковика лабораторних тварин, зокрема білих щурів. Метод дозволяє паралельно визначати різні групи мікроорганізмів у одній пробі вмісту кишковика та за 4 доби отримати повну картину стану мікробоценозу, що дає змогу судити про здоров’я тварин, дисбіотичні порушення у складі мікрофлори кишковика та корекцію їх пробіотичними препаратами. Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine 2015-12-29 Article Article Рецензована Стаття application/pdf application/pdf https://smic.in.ua/index.php/journal/article/view/137 10.35868/1997-3004.22.60-65 Agricultural microbiology; Vol. 22 (2015): Agriciltural microbiology; 60-65 Сільськогосподарська мікробіологія; Том 22 (2015): Сільськогосподарська мікробіологія; 60-65 1997-3004 10.35868/1997-3004.22 en uk https://smic.in.ua/index.php/journal/article/view/137/160 https://smic.in.ua/index.php/journal/article/view/137/161 Авторське право (c) 2015 M. V. Kaminska https://creativecommons.org/licenses/by/4.0 |
| spellingShingle | метод мікрофлора кишковика щурі Камінська, М. В. КОМПЛЕКСНИЙ МЕТОД ВИЗНАЧЕННЯ СКЛАДУ МІКРОФЛОРИ КИШКОВИКА ЛАБОРАТОРНИХ ТВАРИН |
| title | КОМПЛЕКСНИЙ МЕТОД ВИЗНАЧЕННЯ СКЛАДУ МІКРОФЛОРИ КИШКОВИКА ЛАБОРАТОРНИХ ТВАРИН |
| title_alt | THE COMPLEX METHOD FOR DETERMINATION OF INTESTINAL MICROFLORA COMPOSITION OF LABORATORY ANIMALS |
| title_full | КОМПЛЕКСНИЙ МЕТОД ВИЗНАЧЕННЯ СКЛАДУ МІКРОФЛОРИ КИШКОВИКА ЛАБОРАТОРНИХ ТВАРИН |
| title_fullStr | КОМПЛЕКСНИЙ МЕТОД ВИЗНАЧЕННЯ СКЛАДУ МІКРОФЛОРИ КИШКОВИКА ЛАБОРАТОРНИХ ТВАРИН |
| title_full_unstemmed | КОМПЛЕКСНИЙ МЕТОД ВИЗНАЧЕННЯ СКЛАДУ МІКРОФЛОРИ КИШКОВИКА ЛАБОРАТОРНИХ ТВАРИН |
| title_short | КОМПЛЕКСНИЙ МЕТОД ВИЗНАЧЕННЯ СКЛАДУ МІКРОФЛОРИ КИШКОВИКА ЛАБОРАТОРНИХ ТВАРИН |
| title_sort | комплексний метод визначення складу мікрофлори кишковика лабораторних тварин |
| topic | метод мікрофлора кишковика щурі |
| topic_facet | method intestinal microflora rats метод мікрофлора кишковика щурі |
| url | https://smic.in.ua/index.php/journal/article/view/137 |
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