Download LAB 3-7大分子物质的水解实验和IMVIC实验

实验三－七
大分子物质的水解实验和IMVIC实验
一、教学要求
了解微生物一些生化反应的原理及其在微生物鉴
定中的应用。
二、简介
To identify bacteria, we must rely heavily on
biochemical testing. The types of biochemical
reactions each organism undergoes act as a
"thumbprint" for its identification. This is based on
the following chain of logic:
•Each different species of bacterium has a different
molecule of DNA (i.e., DNA with a unique series
of nucleotide bases).
•Since DNA codes for protein synthesis, then
different species of bacteria must, by way of their
unique DNA, be able to synthesize different
protein enzymes.
•Enzymes catalyze all the various chemical
reactions of which the organism is capable. This in
turn means that different species of bacteria must
carry out different and unique sets of biochemical
reactions.
When identifying a suspected organism, you
inoculate a series of differential media After
incubation, you then observe each medium to see
if specific end products of metabolism are
present. This can be done by adding indicators to
the medium that react specifically with the end
product being tested, giving some form of visible
reaction such as a color change. The results of
these tests on the suspected microorganism are
then compared to known results for that organism
to confirm its identification.
1、Macromolecules hydrolysis
•Exoenzymes
diastase（amylase）、protease、 lipase
• endoenzymes
• Starch hydrolysis
Some bacteria are capable of using starch as a source of
carbohydrate but in order to do this, they must first
hydrolyze or break down the starch so it may enter the
cell. The bacterium secretes an exoenzyme which
hydrolyzes the starch by breaking the bonds between the
glucose molecules. This enzyme is called a diastase
（amylase）.
Starch hydrolysis
•protein hydrolysis
Proteins are made up of various amino acids
linked together in long chains by means of
peptide bonds. Many bacteria can hydrolyze a
variety of proteins into peptides (short chains of
amino acids) and eventually into individual
amino acids. They can then use these amino
acids to synthesize their own proteins and other
cellular molecules or to obtain energy. The
hydrolysis of protein is termed proteolysis and
the enzyme involved is called a protease.
protein hydrolysis
2、fermentation of carbohydrates
Facultative anaerobic and anaerobic bacteria are capable of
fermentation, an anaerobic process during which carbohydrates are
broken down for energy production. A wide variety of carbohydrates
may be fermented by various bacteria in order to obtain energy and
the types of carbohydrates which are fermented by a specific
organism can serve as a diagnostic tool for the identification of that
organism.
We can detect whether a specific carbohydrate is fermented by
looking for common end products of fermentation. When
carbohydrates are fermented as a result of bacterial enzymes, the
following fermentation end products may be produced:
• acid end products.
• acid and gas end products.
In order to test for these fermentation products, you
inoculate and incubate tubes of media containing a single
carbohydrate (such as lactose or maltose), a pH
indicator (such as phenol red) and a durham tube (a
small inverted tube to detect gas production). If the
particular carbohydrate is fermented by the bacterium,
acid end products will be produced which lowers the pH,
causing the pH indicator to change color (phenol red
turns yellow) .If gas is produced along with the acid, it
collects in the durham tube as a gas bubble.
3 IMViC and hydrogen sulfide production
• Indole test
•Methyl red test
•Voges-Proskauer test
• Citrate test
•Hydrogen sulfide (H2S) test
• Indole test
Indole is a component of the amino acid
tryptophan. Some bacteria have the ability
to break down tryptophan for nutritional
needs using the enzyme tryptophanase.
When tryptophan is broken down, the
presence of indole can be detected through
the use of Kovacs' reagent. Kovac's
reagent, which is yellow, reacts with indole
and produces a red color on the surface of
the test tube.
• Methyl Red (MR) Test
The combination medium used for this test,
MR/VP broth, includes peptone, glucose, and a
phosphate buffer. Bacteria that are able to perform
a mixed-acid fermentation of glucose and produce
large amounts of stable acids. The pH indicator,
methyl red, is added to a 48 hour culture. If the pH
is less than 4.4, the indicator will turn red. A red
color is read as positive, a yellow color (pH greater
than 6.0) is negative, and an orange color,
indicating a pH between the two, will usually
require further incubation.
The Methyl Red Test: Left to Right:
positive, positive, negative, control.
• Voges-Proskauer (VP) Test
The Voges-Proskauer test uses the same MR/VP
broth. Some fermentative organisms do not
produce enough stable acids to lower the pH of the
medium. For these organisms, the chief end
products of glucose metabolism are acetoin and
2,3-butanediol. After 48 hours of incubation,
Barritt's Reagent A (alpha-napthol) and Barritt's
Reagent B (potassium hydroxide) are added to the
sample. After gently shaking the tube for aeration,
formation of a red color will indicate a positive
reaction. No color change or a copper color are
negative results.
Voges-Proskauer Test
Left: uninoculated control
Right: negative (copper color)
Left: uninoculated control
Right: positive (red color)
• Citrate Utilization Test
citrate agar is a defined medium containing sodium
citrate as the sole carbon source and the ammonium
ion as the sole nitrogen source. The pH indicator,
bromthymol blue, will turn from green at neutral pH
(6.9) to blue when a pH higher than 7.6 is reached
(basic or alkaline). If the citrate is utilized, the
resulting gowth will produce alkaline products (pH
>7.6), changing the color of the medium from green to
blue.
Citrate Utilization
Enterobacter cloacae: positive
Eschericia coli: negative
Klebsiella pneumoniae: positive
• Hydrogen sulfide (H2S) test
Some bacteria are capable of breaking down sulfur containing
amino acids (cystine, methionine) or reducing inorganic
sulfur-containing compounds (such as sulfite, sulfate, or
thiosulfate) to produce hydrogen sulfide (H2S). This reduced
sulfur may then be incorporated into other cellular amino acids,
or perhaps into coenzymes. The ability of an organism to
reduce sulfur-containing compounds to hydrogen sulfide can
be another test for identifying unknown organisms such as
certain Proteus and Salmonella. To test for hydrogen sulfide
production, a medium with a sulfur-containing compound and
iron salts is inoculated and incubated. If the sulfur is reduced
and hydrogen sulfide is produced, it will combine with the
iron salt to form a visible black ferric sulfide (FeS) in the tube.
三 材料与器材
菌种：枯草芽胞杆菌，大肠杆菌，金黄色葡萄
球菌，普通变形杆菌(Proteus vularis).
 培养基：固体油脂培养基，固体淀粉培养基，
明胶培养基试管，石蕊牛奶试管，尿素琼脂试
管.
 试剂：革兰染色用卢戈氏碘液.
 无菌平板，无菌试管，接种环、接种针等.

四 操作步骤
1、Starch hydrolysis
接种、培养、碘液染色、观察
2、Lactose Fermentation
接种、培养、观察。
3、 Methyl red and Voges-Proskauer tests
Inoculate into the MRVP broth.
AFTER INCUBATION （48 hours ）: Pour 1/3 of the
suspension into a clean nonsterile tube: run the MR
test in the tube with 2/3, and the VP test in the open
tube with 1/3.
for methyl red: Add 6-8 drops of methyl red reagent.
for Voges-Proskauer: Add 12 drops of Barritt's
A(alpha-napthol) , mix, 4 drops of Barritt's B (KOH-potassium hydroxide) , mix. Let sit, undisturbed, for at
least 15 minutes.
4、 Citrate test
接种、培养、观察。
五 注意事项
在进行糖发酵培养基接种时，要注意防止倒置
的小管进入气泡。
 配柠檬酸盐培养基时，要注意调节好pH；另外，
接种时要适当加大接种量。

六 思考题
试设计一种可用于蛋白酶产生菌株初筛的筛选
模型。
 产气肠杆菌也可发酵葡萄糖产生有机酸，但其
甲基红反应却为阴性。试分析其原因。
