Análisis Microbiológico. - Recuento de Hongos y Levaduras en placa por siembra en superficie. — Transcript

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1. 0:08Alright, now we are going to proceed
2. 0:09with the inoculation of molds and
3. 0:10yeasts. Yes, in this case we are going
4. 0:12to work with a -1 dilution and a -2
5. 0:14dilution, okay? In duplicate plus the
6. 0:17blank, okay? Or negative control. This,
7. 0:21this plate count is also done uh by
8. 0:24surface inoculation. Right? So, what I
9. 0:28have already done here is label the
10. 0:30sterile plates, add the culture medium,
11. 0:32and wait for it to solidify, because
12. 0:34since it is on the surface, we have to
13. 0:36add the sample uh onto the culture
14. 0:37medium. Right? So, the culture medium
15. 0:42we use is PCA plus chloramphenicol. Yes
16. 0:45, the PCA is a medium, it is the medium
17. 0:48we use for this mesophilic, right? It
18. 0:51is a non-selective culture medium, but
19. 0:53in this case it is combined with
20. 0:55chloramphenicol. Chloramphenicol is an
21. 0:57antibiotic, right? Which allows us to
22. 1:01inhibit the accompanying flora. Yes, we
23. 1:05are looking for molds and yeasts. So,
24. 1:07with that antibiotic what we do is
25. 1:09inhibit the rest of the microorganisms.
26. 1:11Yes, that is why it is used this way.
27. 1:13You can also use PDA or Sabouraud for
28. 1:16molds and yeasts, I mean, there are
29. 1:18other culture media. We use this one
30. 1:21and, according to APHA, it tells us to
31. 1:23use PCA plus chloramphenicol, which is
32. 1:26the most recommended. So, we already
33. 1:29have it solidified. Since it is on the
34. 1:33surface, the inoculation volume is 0.1
35. 1:35ml. So, we are going to take from here
36. 1:38for the -1 dilution. Yes, I already set
37. 1:42the automatic pipette to 0.1 ml, and
38. 1:44now we are going to take it, as it is
39. 1:47in duplicate, meaning it is the same.
40. 1:50Then, once we add the 0.1 ml, we use a
41. 1:54spreader. This spreader is a sterile
42. 1:57spreader. Yes, we have to open it from
43. 1:59this top part, never from the bottom,
44. 2:01because this is the part that will be
45. 2:02in contact with the medium and the
46. 2:04sample. So we open it from the top part
47. 2:07. This is sterile and we pass it over
48. 2:09the surface once we have inoculated.
49. 2:11Right? So now we are going to open it
50. 2:15again here and we are going to add 0.1
51. 2:25ml. Since it is in duplicate and it is
52. 2:28the same one, I can use the same tip.
53. 2:30Yes, always on the surface. We always
54. 2:36take from the supernatant. Yes. And we
55. 2:45discard it. Now we have to use the
56. 2:50spreader. So we open it from the top
57. 2:53part. Everything is sterile. We remove
58. 3:06it and since it is the same dilution
59. 3:07and the same culture medium, we can use
60. 3:10the same spreader. So this is lifted
61. 3:13and passed in a circular motion. Right?
62. 3:16We've rotated the spreader over the
63. 3:18entire surface, right? In the middle,
64. 3:21we spread it out well, distributing
65. 3:24that 0.1 ml of sample evenly. We do the
66. 3:28same for the other one. We can use the
67. 3:32same spreader. In this case, I repeat,
68. 3:34because it's the same dilution, the
69. 3:37same sample, and the same culture
70. 3:39medium. Yes. Once it covers everything,
71. 3:43we leave it there. This spreader is now
72. 3:46ready to be discarded. Now we will
73. 3:50proceed to the next dilution, right? We
74. 3:53do the same thing. We set this aside.
75. 3:59And now we do the same for dilution two
76. 4:01. We will take 1 ml. We take another
77. 4:29spreader. And we open plate three. And
78. 4:58we spread it. This one we discard, and
79. 5:25now we do the same for the control. Yes
80. 5:28, the control contains the culture
81. 5:30medium plus the diluent. So, now we do
82. 5:33the same. Here I have the control; here
83. 5:37I have the tube that has only sterile
84. 5:39peptone water. And now we do the same.
85. 5:45And there we rule out that what grows
86. 5:48is really what is there; in other words
87. 5:51, it’s not due to possible
88. 5:53contamination or poor sterilization.
89. 5:57Yes. We discard it. We take another
90. 6:03small spreader. And we spread it. Now
91. 6:30we let it solidify a bit, let the
92. 6:34sample come into good contact with the
93. 6:38medium, and then we take them to
94. 6:41incubate. Molds and yeasts are
95. 6:43incubated this way; they are not
96. 6:45inverted, they go like this. Right,
97. 6:53once we have the molds and yeasts ready
98. 6:55, yes? Now what we do is place them, we
99. 6:58wrap them in paper this way, we don't
100. 7:01invert them; these do go face up as
101. 7:04they are. Yes, we place them here. We
102. 7:20stack them well. Yes. Date it was
103. 7:24seeded, date it needs to be taken out,
104. 7:27and what organism it is. You can also
105. 7:32include the temperature for them. Yes.
106. 7:37And molds and yeasts are at 25 degrees
107. 7:41for 5 days. Yes. So now it goes into
108. 7:48the incubator at 25 degrees. 5 days. M.